Fatty acids and their use in conjugation to biomolecules

ABSTRACT

The invention provides a conjugate comprising a biomolecule linked to a fatty acid via a linker wherein the fatty acid has the following Formulae A1, A2 or A3: 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 1 , R 2 , R 3 , R 4 , Ak, n, m and p are defined herein. The invention also relates to a method for manufacturing the conjugate of the invention such as GDF15 conjugate, and its therapeutic uses such as treatment or prevention of metabolic disorders or diseases, type 2 diabetes mellitus, obesity, pancreatitis, dyslipidemia, alcoholic and nonalcoholic fatty liver disease/steatohepatitis and other progressive liver diseases, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, hypertension, cardiovascular disease, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, diabetic complications (including but not limited to chronic kidney disease), neuropathy, gastroparesis and other metabolic disorders. The present invention further provides a combination of pharmacologically active agents and a pharmaceutical composition.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/738,272, filed on Jun. 12, 2015, which claims priority to, andthe benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.62/107,016, filed Jan. 23, 2015, U.S. Provisional Application No.62/082,327, filed on Nov. 20, 2014, and U.S. Provisional Application No.62/015,862 filed on Jun. 23, 2014, the entire contents of each of whichare incorporated herein by reference in their entireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “PAT056274-US-DIV_SequenceListing_ST25.K” which was created on May 21, 2018 and is 33 KB in size,are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to novel conjugates of GDF15 which haveimproved half-life and duration of action, method of making them andusing them. The invention further relates to novel fatty acids and theiruse in extending the half-life of biomolecules via conjugation.

BACKGROUND OF THE INVENTION

Peptides and proteins are widely used in medical practice, and sincethey can be produced by recombinant DNA technology it can be expectedthat their importance will increase also in the years to come. Thenumber of known endogenous peptides and proteins with interestingbiological activities is growing rapidly, also as a result of theongoing exploration of the human genome. Due to their biologicalactivities, many of these polypeptides and proteins could in principlebe used as therapeutic agents. Endogenous peptides or proteins are,however, not always suitable as drug candidates because they often havehalf-lives of few minutes due to rapid degradation by peptidases and/ordue to renal filtration and excretion in the urine. The half-life ofpolypeptides or proteins in human plasma varies strongly (from a fewminutes to more than one week).

A high clearance of a therapeutic agent is inconvenient in cases whereit is desired to maintain a high blood level thereof over a prolongedperiod of time. One way which has been currently used to overcome thisdisadvantage is to administer large dosage of therapeutic peptide orproteins of interest to the patient so that even if some therapeuticpeptide or protein is degraded, enough remains to be therapeuticallyeffective. However, this method is uncomfortable to patients. Since mosttherapeutic peptides or proteins cannot be administered orally, thetherapeutic peptide or proteins would have to be either constantlyinfused, frequently infused by intravenous injection or administeredfrequently by the inconvenient route of subcutaneous injections. Theneed for frequent administration also results in much potential peptideor protein therapeutics having an unacceptable high projected cost oftreatment. The presence of large amounts of degraded peptide or proteinmay also generate undesired side effects.

Discomfort in administration and high costs are two reasons why mosttherapeutic peptides or proteins with attractive bioactivity profilesmay not be developed as drug candidates.

Therefore, one approach to prolong half-life of peptides or proteins isto modify the therapeutic peptides or proteins in such a way that theirdegradation is slowed down while still maintaining biological activity.Serum albumin has a half-life of more than one week, and one approach toincreasing the plasma half-life of peptides or proteins has been toderivatize them with a chemical entity that binds to serum albumin orother plasma proteins.

However, there is still a need to identify new half-life extendingmoieties to modify therapeutic biomolecules such as peptides andproteins in order to provide longer duration of action in vivo whilemaintaining low toxicity and therapeutic advantages.

SUMMARY OF THE INVENTION

The present invention relates to a conjugate comprising a biomoleculelinked to a fatty acid via a linker wherein the fatty acid has thefollowing Formulae A1, A2 or A3:

R¹ is CO₂H or H;R², R³ and R⁴ are independently of each other H, OH, CO₂H, —CH═CH₂ or—C═CH;Ak is a branched C₆-C₃₀alkylene;n, m and p are independently of each other an integer between 6 and 30,or an amide, an ester or a pharmaceutically acceptable salt thereof.

The fatty acid of Formulae A1, A2 and A3 when conjugated to abiomolecule of interest via a linker have been found to increase thehalf-life of said biomolecule to a much greater extent than morecommonly used fatty acid residues.

In another embodiment, the invention pertains to a conjugate comprisinga biomolecule liked to a fatty acid of Formulae A1 wherein at least oneof R² and R³ is CO2H.

In another embodiment of the present invention, the biomolecule ofinterest is a therapeutic peptide, a therapeutic protein or a RNA. Inyet another aspect of this embodiment, the biomolecule of interest is apeptide or polypeptide. In yet a further aspect of this embodiment, thepeptide or polypeptide is an APJ agonist peptide, an oxytocin receptoragonist peptide, serelaxin, NPFF, a PIP peptide, an FGF23 peptide, anAgRP peptide or a Growth Differentiation Factor 15 (GDF15) protein,homologs, variants, fragments and other modified forms thereof.

In yet another embodiment, the invention pertains to pharmaceuticalcompositions, comprising a conjugate of the invention and one or morepharmaceutically acceptable carriers.

In still another embodiment, the invention pertains to combinationsincluding, a conjugate of the invention, and pharmaceutical combinationsof one or more therapeutically active agents.

In another embodiment, the invention pertains to the fatty acid ofFormulae A1, A2 or A3.

In another embodiment, the present invention contemplates the use of theconjugates described herein, and composition thereof, to treat and/orprevent various diseases, disorders and conditions, and/or the symptomsthereof.

For example, the invention pertains to a method for activation of theAPJ receptor in a subject in need thereof, comprising: administering tothe subject a therapeutically effective amount of a conjugate of theinvention wherein the biomolecule is an APJ agonist. The conjugates ofthe invention, via activation of the APJ receptor, have utility in thetreatment of acute decompensated heart failure (ADHF), chronic heartfailure, pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.In a preferred aspect of this embodiment the conjugates of the inventionare useful in the treatment of acute decompensated heart failure (ADHF)or chronic heart failure.

In another embodiment, the invention pertains to a method for activationof the oxytocin receptor in a subject in need thereof; comprising:administering to the subject a therapeutically effective amount of aconjugate of the invention wherein the biomolecule is an oxytocinreceptor agonist peptide. The conjugates of the invention, viaactivation of the oxytocin receptor, have utility in the treatment ofautism (therapeutic or prophylactic), migraine, attention deficithyperactivity disorder (ADHD), oppositional defiant disorder (ODD),stress, including post traumatic stress disorder, anxiety, includinganxiety disorders and depression, schizophrenia, psychiatric disordersand memory loss, alcohol withdrawal, drug addiction, Prader-WilliSyndrome, metabolic disorders or diseases, type 2 diabetes mellitus,obesity, dyslipidemia, elevated glucose levels, elevated insulin levelsand diabetic nephropathy, fibromyalgia, sleep disorder, sleep apnea,diastolic heart failure, urine incontinence, atherosclerosis,hypertension, erectile dysfunction, prostatic hypertrophy symptoms,non-alcoholic fatty liver disease, compromised lactation conditions,labor induction impairment, uterine atony conditions, excessivebleeding, inflammation, pain, abdominal pain, back pain, male and femalesexual dysfunction, irritable bowel syndrome (IBS), constipation,gastrointestinal obstruction, surgical blood loss, postpartumhaemorrhage, wound healing, infection, mastitis, placenta deliveryimpairment, osteoporosis; and for the diagnosis of cancer and placentalinsufficiency. In a preferred aspect of this embodiment the conjugatesof the invention are useful in the treatment of autism, anxiety,including anxiety disorders and depression, Migraine, ADHD, OppositionalDefiant Disorder, schizophrenia, psychiatric disorders, obesity,compromised lactation conditions, labor induction impairment, uterineatony conditions, excessive bleeding, postpartum hemorrhage. Yet in amore preferred embodiment, the conjugates of the invention are usefulfor the treatment of Prader-Willi Syndrome.

In yet another embodiment, the invention pertains to a method oftreating Cushing's syndrome, Hypercortisolism, the ectopic ACTHsyndrome, the change in adrenocortical mass, primary pigmented nodularadrenocortical disease (PPNAD) Carney complex (CNC), thecortisol-induced mineralocorticoid excess, Conditions associated withpost-traumatic stress disorder, hirsutism, thin skin, myopathy,osteoporosis, increased tissue fragility, poor wound healing,hypertension, diabetes mellitus, low serum potassium, low eosinophilsand lymphopenia, comprising: administering to the subject atherapeutically effective amount of a conjugate of the invention whereinthe biomolecule is an AgRP peptide. In a preferred aspect of thisembodiment the conjugates of the invention are useful in the treatmentof Cushing's syndrome, Hypercortisolism, the ectopic ACTH syndrome,osteoporosis.

In yet another embodiment, the invention pertains to a method oftreating or preventing FGF23-related diseases such as age-relatedconditions (selected from the group consisting of sarcopenia, skinatrophy, muscle wasting, brain atrophy, atherosclerosis,arteriosclerosis, pulmonary emphysema, osteoporosis, osteoarthritis,immunologic incompetence, high blood pressure, dementia, Huntington'sdisease, Alzheimer's disease, cataracts, age-related maculardegeneration, prostate cancer, stroke, diminished life expectancy,memory loss, wrinkles, impaired kidney function, and age-related hearingloss), a metabolic disorder (selected from the group consisting of TypeII Diabetes, Metabolic Syndrome, hyperglycemia, and obesity),hyperphosphatemia (tumoral calcinosis, hyperphosphatemic hyperostosissyndrome), chronic renal disease, chronic renal failure, cancer, breastcancer, and/or muscle atrophy; comprising: administering to the subjecta therapeutically effective amount of a conjugate of the inventionwherein the biomolecule is a FGF23 peptide.

In yet another embodiment, the invention pertains to a method oftreating acute heart failure, chronic heart failure with reducedejection fraction (HFrEF), chronic heart failure with preserved ejectionfraction (HFpEF), diastolic dysfunction, post myocardial remodeling,angina, hypertension, pulmonary hypertension, pulmonary arteryhypertension, fibrosis (diffuse, cardiac, renal, pulmonary, liver),scleroderma, wound healing, critical limb ischemia, peripheral vasculardisease, intermittent claudication, renal dysfunction and chronic kidneydisease; comprising: administering to the subject a therapeuticallyeffective amount of a conjugate of the invention wherein the biomoleculeis a serelaxin peptide. In a preferred aspect of this embodiment, theserelaxin conjugates of the invention are useful in the treatment ofacute heart failure, chronic heart failure with reduced ejectionfraction (HFrEF), chronic heart failure with preserved ejection fraction(HFpEF), diastolic dysfunction, post myocardial remodeling, angina,hypertension, pulmonary hypertension or pulmonary artery hypertension.

In yet another embodiment, the invention pertains to a method oftreating or preventing metabolic disorders such as type 2 diabetesmellitus (T2DM), pancreatic beta cell impairment, glucose intolerance,hyperglycemia, insulin resistance, obesity, dyslipidemia, nonalcoholicsteatohepatitis (NASH), metabolic syndrome, and other metabolicdisorders; comprising: administering to the subject a therapeuticallyeffective amount of a conjugate of the invention wherein the biomoleculeis a PIP peptide.

In yet another embodiment, the invention pertains to a method oftreating or preventing metabolic disorders or diseases, type 2 diabetesmellitus, obesity, pancreatitis, dyslipidemia, nonalcoholicsteatohepatitis, insulin resistance, hyperinsulinemia, glucoseintolerance, hyperglycemia, metabolic syndrome, hypertension,cardiovascular disease, atherosclerosis, peripheral arterial disease,stroke, heart failure, coronary heart disease, diabetic complications(including but not limited to chronic kidney disease), neuropathy,gastroparesis, urge incontinence, sedation, neuropathic and inflammatorypain, memory loss, and other metabolic disorders; comprising:administering to the subject a therapeutically effective amount of aconjugate of the invention wherein the biomolecule is NPFF peptide.

In still another aspect of the present invention, the invention pertainsto a method of treating metabolic disorders or diseases, diabetes, type2 diabetes mellitus, obesity, alcoholic and nonalcoholic fatty liverdisease/steatohepatitis and other progressive liver diseases,pancreatitis, dyslipidemia, insulin resistance, hyperinsulinemia,glucose intolerance, hyperglycemia, metabolic syndrome, hypertension,cardiovascular disease, atherosclerosis, peripheral arterial disease,stroke, heart failure, coronary heart disease, diabetic complications(including but not limited to chronic kidney disease), neuropathy,gastroparesis and other metabolic disorders, in a subject in needthereof, comprising: administering to the subject a therapeuticallyeffective amount of a conjugate of the invention wherein the biomoleculeis human Growth Differentiation Factor 15 (GDF15), homologs, variants,mutants, fragments and other modified forms thereof.

These and other aspects of the invention will be elucidated in thefollowing detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows that Example 24 decreased plasma APOC3 levels moreeffectively than reference example 3.

DETAILED DESCRIPTION Definition

For purposes of interpreting this specification, the followingdefinitions will apply unless specified otherwise and wheneverappropriate, terms used in the singular will also include the plural andvice versa.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “theconjugate” includes reference to one or more conjugates; reference to“the polypeptide” includes reference to one or more polypeptides; and soforth.

The term alkyl refers to a fully saturated branched or unbranched (orstraight chain or linear) hydrocarbon moiety, comprising 1 to 30 carbonatoms. Preferably the alkyl comprises 5 to 20 carbon atoms, and morepreferably 10 to 15 carbon atoms. C₁₀₋₁₅alkyl refers to an alkyl chaincomprising 10 to 15 carbons. The term “alkylene” refer to a divalentalkyl as defined supra.

The term “alkenyl” refers to a branched or unbranched hydrocarbon havingat least one carbon-carbon double bond. The term “C₂₋₃₀-alkynyl” refersto a hydrocarbon having two to seven carbon atoms and comprising atleast one carbon-carbon triple

The term “alkynyl” refers to a branched or unbranched hydrocarbon havingat least one carbon-carbon triple bond. The term “C₂₋₃₀-alkynyl” refersto a hydrocarbon having two to seven carbon atoms and comprising atleast one carbon-carbon triple bond.

The term aryl refers to monocyclic or bicyclic aromatic hydrocarbongroups having 6-10 carbon atoms in the ring portion. Representativeexamples of aryl are phenyl or naphthyl.

The term heteroaryl includes monocyclic or bicyclic heteroaryl,containing from 5-10 ring members selected from carbon atoms and 1 to 5heteroatoms, and each heteroatoms is independently selected from O, N orS wherein S and N may be oxidized to various oxidation states. Forbicyclic heteroaryl system, the system is fully aromatic (i.e. all ringsare aromatic).

The term cycloalkyl refers to saturated or unsaturated but non-aromaticmonocyclic, bicyclic or tricyclic hydrocarbon groups of 3-12 carbonatoms, preferably 3-8, or 3-7 carbon atoms. For bicyclic, and tricycliccycloalkyl system, all rings are non-aromatic. For example, cycloalkylencompasses cycloalkenyl and cycloalkynyl. The term “cycloalkenyl”refers to a bicyclic or tricyclic hydrocarbon group of 3-12 carbonatoms, having at least one carbon-carbon double bond. The term“cycloalkynyl” refers to a bicyclic or tricyclic hydrocarbon group of3-12 carbon atoms, having at least one carbon-carbon triple bond.

The term heterocyclyl refers to a saturated or unsaturated non-aromatic(partially unsaturated but non-aromatic) monocyclic, bicyclic ortricyclic ring system which contains at least one heteroatom selectedfrom O, S and N, where the N and S can also optionally be oxidized tovarious oxidation states. In one embodiment, heterocyclyl moietyrepresents a saturated monocyclic ring containing from 5-7 ring atomsand optionally containing a further heteroatom, selected from O, S or N.The heterocyclic ring may be substituted with alkyl, halo, oxo, alkoxy,haloalkyl, haloalkoxy. In other embodiment, heterocyclyl is di- ortricyclic. For polycyclic system, some ring may be aromatic and fused tosaturated or partially saturated ring or rings. The overall fused systemis not fully aromatic. For example, a heterocyclic ring system can be anaromatic heteroaryl ring fused with saturated or partially saturatedcycloalkyl ring system.

The term “conjugate” is intended to refer to the entity formed as aresult of a covalent attachment of biomolecule and a fatty acid moiety,via a linker. The term “conjugation” refers to the chemical reactionresulting in the covalent attachment of the biomolecule and the fattyacid moiety.

Biomolecule:

As used herein the term biomolecule includes, but is not limited to,antibodies (e.g., monoclonal, chimeric, humanized, nanobodies, andfragments thereof etc.), cholesterol, hormones, peptides, proteins,chemotherapeutics and other types of antineoplastic agents, lowmolecular weight drugs, vitamins, co-factors, nucleosides, nucleotides,oligonucleotides, enzymatic nucleic acids, antisense nucleic acids,triplex forming oligonucleotides, antisense DNA or RNA compositions,chimeric DNA:RNA compositions, allozymes, aptamers, ribozyme, decoys andanalogs thereof, plasmids and other types of expression vectors, andsmall nucleic acid molecules, RNAi agents, short interfering nucleicacid (siNA), messenger ribonucleic acid” (messenger RNA, mRNA), shortinterfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA),and short hairpin RNA (shRNA) molecules, peptide nucleic acid (PNA), alocked nucleic acid ribonucleotide (LNA), morpholino nucleotide, threosenucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internallysegmented interfering RNA), aiRNA (assymetrical interfering RNA), andsiRNA with 1, 2 or more mismatches between the sense and anti-sensestrand to relevant cells and/or tissues, such as in a cell culture,subject or organism. Such compounds may be purified or partiallypurified, and may be naturally occurring or synthetic, and may bechemically modified.

In one embodiment the biomolecule is a polypeptide, peptide, proteins ora RNAi agent, short interfering nucleic acid (siNA), short interferingRNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), or a shorthairpin RNA (shRNA) molecule.

In other embodiment the biomolecule is a polypeptide (or protein), orpeptide. Examples of polypeptides or peptides are APJ agonist peptides,oxytocin peptides, serelaxin, NPFF, a PIP peptide, an FGF23 peptide,AgRP peptides and GDF15 peptide. In a preferred embodiment, thebiomolecule is GDF15 polypeptide, homolog, variant, mutant, fragment andother modified forms thereof.

A “ribonucleic acid” (RNA) is a polymer of nucleotides linked by aphosphodiester bond, where each nucleotide contains ribose or amodification thereof as the sugar component. Each nucleotide contains anadenine (A), a guanine (G), a cytosine (C), a uracil (U) or amodification thereof as the base. The genetic information in a mRNAmolecule is encoded in the sequence of the nucleotide bases of the mRNAmolecule, which are arranged into codons consisting of three nucleotidebases each. Each codon encodes for a specific amino acid of thepolypeptide, except for the stop codons, which terminate translation(protein synthesis). Within a living cell, mRNA is transported to aribosome, the site of protein synthesis, where it provides the geneticinformation for protein synthesis synthesis (translation). For a fullerdescription, see, Alberts B et al. (2007) Molecular Biology of the Cell,Fifth Edition, Garland Science.

The terms “RNAi agent,” “short interfering RNA”, “siRNA”, “shortinterfering nucleic acid”, “siNA” and the like as used herein refers toany nucleic acid molecule capable of inhibiting or down regulating geneexpression or viral replication by mediating RNA interference (RNAi) orgene silencing in a sequence-specific manner. The terms include shortinterfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA),short interfering oligonucleotides, chemically-modified shortinterfering nucleic acid molecules, sisiRNA (small internally segmentedinterfering RNA), aiRNA (assymetrical interfering RNA), siRNA with 1, 2or more mismatches between the sense and anti-sense strand to relevantcells and/or tissues, RNAi agents wherein one or more mismatches existbetween the sense and antisense strands, RNAi agents wherein the sensestrand is very short relative to the antisense strand and/or has one ormore single-stranded nicks, or any other molecule capable of mediatingRNA interference. RNAi agents can comprise ribonucleotides, or bemodified or substituted at one or more sugar, base and/or phosphate. Asnon-limiting examples, the RNAi agents can be modified at the 2′position with a 2′-modification selected from the group consisting of:2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), and 2′-O—N-methylacetamido (2′-O-NMA). In one embodiment,all pyrimidines (uridine and cytidine) are 2′-O-methyl-modifiednucleosides. In various embodiments, one or more phosphate can besubstituted with a modified internucleoside linker, selected fromphosphorothioate, phosphorodithioate, phosphoramidate,boranophosphonoate, and an amide linker. In various embodiments, one ormore nucleotides can be modified, or substituted with DNA, a peptidenucleic acid (PNA), locked nucleic acid (LNA), morpholino nucleotide,threose nucleic acid (TNA), glycol nucleic acid (GNA), arabinose nucleicacid (ANA), 2′-fluoroarabinose nucleic acid (FANA), cyclohexene nucleicacid (CeNA), anhydrohexitol nucleic acid (HNA), unlocked nucleic acid(UNA). Various modifications and substitutions of RNAi agents are knownin the art and can be used in the context of the instant disclosure.siRNAs are responsible for RNA interference, the process ofsequence-specific post-transcriptional gene silencing in animals andplants. siRNAs are generated naturally by ribonuclease III cleavage fromlonger double-stranded RNA (dsRNA) which are homologous to, or specificto, the silenced gene target; artificial RNAi agents can be produced byany method known in the art.

As used herein, the term “polypeptide” refers to a polymer of amino acidresidues linked by peptide bonds, whether produced naturally orsynthetically. Polypeptides of less than about 10 amino acid residuesare commonly referred to as “peptides.” The term “peptide” is intendedto indicate a sequence of two or more amino acids linked by peptidebonds, wherein said amino acids may be natural or unnatural. The termencompasses the terms polypeptides and proteins, which may consist oftwo or more peptides held together by covalent interactions, such as forinstance cysteine bridges, or non-covalent interactions. Theart-recognized three letter or one letter abbreviations are used torepresent amino acid residues that constitute the peptides andpolypeptides of the invention. Except when preceded with “D”, the aminoacid is an L-amino acid. When the one letter abbreviation is a capitalletter, it refers to the D-amino acid. When the one letter abbreviationis a lower case letter, it refers to the L-amino acid. Groups or stringsor amino acid abbreviations are used to represent peptides. Peptides areindicated with the N-terminus on the left and the sequence is writtenfrom the N-terminus to the C-terminus.

Peptides of the invention contain non-natural amino acids (i.e.,compounds that do not occur in nature) and other amino acid analogs asare known in the art may alternatively be employed.

Certain non-natural amino acids can be introduced by the technologydescribed in Deiters et al., J Am Chem Soc 125:11782-11783, 2003; Wangand Schultz, Science 301:964-967, 2003; Wang et al., Science292:498-500, 2001; Zhang et al., Science 303:371-373, 2004 or in U.S.Pat. No. 7,083,970. Briefly, some of these expression systems involvesite-directed mutagenesis to introduce a nonsense codon, such as anamber TAG, into the open reading frame encoding a polypeptide of theinvention. Such expression vectors are then introduced into a host thatcan utilize a tRNA specific for the introduced nonsense codon andcharged with the non-natural amino acid of choice. Particularnon-natural amino acids that are beneficial for purpose of conjugatingmoieties to the polypeptides of the invention include those withacetylene and azido side chains.

A “protein” is a macromolecule comprising one or more polypeptidechains. Each of those polypeptide chains may be conjugated with a fattyacid molecule of Formula A1, A2 or A3. A protein may also comprisenon-peptidic components, such as carbohydrate groups. Carbohydrates andother nonpeptidic substituents may be added to a protein by the cell inwhich the protein is produced, and will vary with the type of cell.Proteins are defined herein in terms of their amino acid backbonestructures; substituents such as carbohydrate groups are generally notspecified, but may be present nonetheless. A protein or polypeptideencoded by a non-host DNA molecule is a “heterologous” protein orpolypeptide.

An “isolated polypeptide or isolated protein” is a polypeptide orprotein (for example GDF15) that is essentially free from cellularcomponents, such as carbohydrate, lipid, or other proteinaceousimpurities associated with the polypeptide in nature. Typically, apreparation of isolated polypeptide or protein contains the polypeptideor protein in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure,such as 96%, 97%, or 98% or more pure, or greater than 99% pure. One wayto show that a particular protein preparation contains an isolatedpolypeptide or protein is by the appearance of a single band followingsodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of theprotein preparation and Coomassie Brilliant Blue staining of the gel.However, the term “isolated” does not exclude the presence of the samepolypeptide or protein in alternative physical forms, such as dimers oralternatively glycosylated or derivatized forms. Preferably, theisolated polypeptide is substantially free from any other contaminatingpolypeptides or other contaminants that are found in its naturalenvironment that would interfere with its therapeutic, diagnostic,prophylactic or research use.

One of ordinary skill in the art will appreciate that various amino acidsubstitutions, e.g, conservative amino acid substitutions, may be madein the sequence of any of the polypeptide or protein described herein,without necessarily decreasing its activity. As used herein, “amino acidcommonly used as a substitute thereof” includes conservativesubstitutions (i.e., substitutions with amino acids of comparablechemical characteristics). For the purposes of conservativesubstitution, the non-polar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophanand methionine. The polar (hydrophilic), neutral amino acids includeserine, threonine, cysteine, tyrosine, asparagine, and glutamine. Thepositively charged (basic) amino acids include arginine, lysine andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid. Examples of amino acid substitutions includesubstituting an L-amino acid for its corresponding D-amino acid,substituting cysteine for homocysteine or other non-natural amino acidshaving a thiol-containing side chain, substituting a lysine forhomolysine, diaminobutyric acid, diaminopropionic acid, ornithine orother non-natural amino acids having an amino containing side chain, orsubstituting an alanine for norvaline or the like.

The term “amino acid,” as used herein, refers to naturally occurringamino acids, unnatural amino acids, amino acid analogues and amino acidmimetics that function in a manner similar to the naturally occurringamino acids, all in their D and L stereoisomers if their structureallows such stereoisomeric forms. Amino acids are referred to herein byeither their name, their commonly known three letter symbols or by theone-letter symbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission.

The term “naturally occurring” refers to materials which are found innature and are not manipulated by man. Similarly, “non-naturallyoccurring,” “un-natural,” and the like, as used herein, refers to amaterial that is not found in nature or that has been structurallymodified or synthesized by man. When used in connection with aminoacids, the term “naturally occurring” refers to the 20 conventionalamino acids (i.e., alanine (A or Ala), cysteine (C or Cys), asparticacid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe),glycine (G or Gly), histidine (H or His), isoleucine (I or lie), lysine(K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N orAsn), proline (P or Pro), glutamine (Q or Gin), arginine (R or Arg),serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan(W or Trp), and tyrosine (Y or Tyr)).

The terms “non-natural amino acid” and “unnatural amino acid,” as usedherein, are interchangeably intended to represent amino acid structuresthat cannot be generated biosynthetically in any organism usingunmodified or modified genes from any organism, whether the same ordifferent. The terms refer to an amino acid residue that is not presentin the naturally occurring (wild-type) protein sequence or the sequencesof the present invention. These include, but are not limited to,modified amino acids and/or amino acid analogues that are not one of the20 naturally occurring amino acids, selenocysteine, pyrrolysine (Pyl),or pyrroline-carboxy-lysine (Pcl, e.g., as described in PCT patentpublication WO2010/48582). Such non-natural amino acid residues can beintroduced by substitution of naturally occurring amino acids, and/or byinsertion of non-natural amino acids into the naturally occurring(wild-type) protein sequence or the sequences of the invention. Thenon-natural amino acid residue also can be incorporated such that adesired functionality is imparted to the molecule, for example, theability to link a functional moiety (e.g., PEG). When used in connectionwith amino acids, the symbol “U” shall mean “non-natural amino acid” and“unnatural amino acid,” as used herein.

The term “analogue” as used herein referring to a polypeptide or proteinmeans a modified peptide or protein wherein one or more amino acidresidues of the peptide/protein have been substituted by other aminoacid residues and/or wherein one or more amino acid residues have beendeleted from the peptide/protein and/or wherein one or more amino acidresidues have been added the peptide/protein. Such addition or deletionof amino acid residues can take place at the N-terminal of the peptideand/or at the C-terminal of the peptide.

As used herein, the term “ester of the conjugate” refers to a conjugatewhich comprises a peptide or polypeptide wherein an ester derivative ofa carboxylic acid group is present (e.g —CO₂H at the C-terminus has beenconverted to —COOR) form wherein R of the ester refers to C₁₋₆ alkylgroups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc., C₃₋₈cycloalkyl groups such as cyclopentyl, cyclohexyl, etc., C₆₋₁₀ arylgroups such as phenyl, α-naphthyl, etc., C₆₋₁₀ aryl-C₁₋₆ alkyl groups,for example phenyl-C₁₋₂ alkyl groups such as benzyl, phenethyl,benzhydryl, etc., and α-naphthyl-C₁₋₂ alkyl groups such asα-naphthylmethyl and the like. When the peptide or polypeptide moiety ofthe conjugate possess additional carboxyl or carboxylate groups inpositions other than the C terminus, those polypeptides in which suchgroups are amidated or esterified also fall under the category of thepolypeptide of the invention. In such cases, the esters may for examplebe the same kinds of esters as the C-terminal esters mentioned above.

As used herein the term “amide of the conjugate” refers to a conjugatewhich comprises a peptide or polypeptide wherein an amide derivative ofa carboxylic acid group is present (e.g., —CO₂H has been converted to—CO(NR′R′) wherein R′ is H or R and R is defined above. The term “amideof the conjugate” also refers to a conjugate which comprises a peptideor polypeptide wherein an amide derivative of an amino group is present(i.e., other than the amino group conjugated to a fatty acid) (e.g.,—NH₂ has been converted to —NH—C(O)R) wherein R is defined supra. In apreferred embodiment, an “amide of the conjugate” is a conjugate whichcomprises a peptide or polypeptide wherein the carboxylic group at theC-terminus has been amidated (e.g., —CO₂H has been converted to—C(O)NH₂, —C(O)NH—C₁₋₆ alkyl, —C(O)NH—C₁₋₂alkylphenyl, or —C(O)N(C₁₋₆alkyl)₂).

The term “APJ” (also referred to as “apelin receptor,”“angiotensin-like-1 receptor,” “angiotensin II-like-1 receptor,” and thelike) indicates a 380 residue, 7 transmembrane domain, Gi coupledreceptor whose gene is localized on the long arm of chromosome 11 inhumans (NCBI Reference Sequence: NP_005152.1, and encoded by NCBIReference Sequence: NM_005161). APJ was first cloned in 1993 fromgenomic human DNA using degenerate oligonucleotide primers (O'Dowd etal. Gene, 136:355-60, 1993) and shares significant homology withangiotensin II receptor type 1. Despite this homology however,angiotensin II does not bind APJ. Although orphan for many years, theendogenous ligand has been isolated and named apelin (Tatemoto et al.,Biochem Biophys Res Commun 251, 471-6 (1998)).

The term “APJ agonist” includes apelin polypeptides: Apelin indicates a77 residue preprotein (NCBI Reference Sequence: NP_0059109.3, andencoded by NCBI Reference Sequence: NM_017413.3), which gets processedinto biologically active forms of apelin peptides, such as apelin-36,apelin-17, apelin-16, apelin-13, apelin-12. The full length maturepeptide, referred to as “apelin-36,” comprises 36 amino acids, but themost potent isoform is the pyroglutamated form of a 13mer of apelin(apelin-13), referred to as “Pyr-1-apelin-13 or Pyr¹-apelin-13”Different apelin forms are described, for instance, in U.S. Pat. No.6,492,324B1. Apelin peptide agonists are also described in patentapplication numbers WO 2013/111110, U.S. application Ser. No.14/082,771, and provisional U.S. application No. 61/858,263, 61/858,280and 61/858,290 which are incorporated by reference herein.

The term “oxytocin receptor agonist peptide” or “oxytocin peptide” areused interchangeably and includes oxytocin and its analogs. Oxytocin isa nine amino acid cyclic peptide hormone with two cysteine residues thatform a disulfide bridge between position 1 and 6. Human oxytocincomprises the sequence Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly (SEQ IDNO:14). The term “oxytocin receptor agonist peptide” also includesanalogs of oxytocin that retain bioactivity. Such analog molecules arecapable of acting in a similar manner to endogenous oxytocin, includingbinding the oxytocin receptor. Analogs of oxytocin of particularinterest are those disclosed in PCT application No. WO 2014/095773(particularly Example 13); those disclosed in US patent application No.US2011/044905 (particularly Example 49); and those disclosed inKazimierz Wisniewski et al., Journal of Medicinal Chemistry 2014, 57,5306-5317 and Zbigniew Grzonka et al., Journal of Medicinal Chemistry1983, 26, 1786-1787; which are all incorporated by reference herein.

By “PIP” or “Prolactin-Inducible Peptide” is meant the protein withGenBank Accession No. NP_002643 that exhibits roles in diversebiological processes. PIP is also known in the art as gross cysticdisease fluid protein-15 (GCDFP-15); secretory actin-binding protein(SABP); extraparotid glycoprotein (EP-GP); and 17-kDa CD4-bindingprotein (GP17). PIP is expressed in exocrine organs, and in benign andmalignant human breast tumors. The mature secreted PIP protein has amolecular mass of 13 kDa and it runs as a 17-20 kDa polypeptide inSDS-PAGE suggesting a glycosylation event. PIP is expressed in mostorgans that contribute to human body fluids; the expression of PIP ishighest in salivary gland followed by lacrimal, prostrate, muscle,trachea and mammary glands. The PIP gene encodes the PIP polypeptide.

By “PIP peptide” as used herein is meant human PIP or a homolog,variant, fragment or modified form thereof, which retains at least oneactivity of human PIP.

The sequence of a non-limiting example of human PIP is presented in SEQID NO: 11:

(SEQ ID NO: 11)   1MRLLQLLFRA SPATLLLVLC LQLGANKAQD NTRKIIIKNF DIPKSVRPND EVTAVLAVQT  61ELKECMVVKT YLISSIPLQG AFNYKYTACL CDDNPKTFYW DFYTNRTVQI AAVVDVIREL 121GICPDDAAVI PIKNNRFYTI EILKVE 

SEQ ID NO: 11 represents the full length human wild-type PIP, includingthe signal peptide (amino acids 1-28), which is not required forfunction.

Another non-limiting example of the term “PIP” as used herein is aminoacids (aa) 29-146 of SEQ ID NO: 11, which thus lacks the signal peptide(amino acids 1-28) and is presented below as SEQ ID NO: 12.

(SEQ ID NO: 12)  1QDNTRKIIIK NFDIPKSVRP NDEVTAVLAV QTELKECMVV KTYLISSIPL QGAFNYKYTA 61CLCDDNPKTF YWDFYTNRTV QIAAVVDVIR ELGICPDDAA VIPIKNNRFY TIEILKVE

By a “homolog,” “variant”, “fragment” or “modified form” of PIP or thelike is meant a polypeptide similar but non-identical to a human PIP,but which retains at least one activity of human PIP. Such a polypeptidecan have a sequence not identical to that of human PIP (e.g., SEQ ID NO:12), or have a sequence identical to that of human PIP (e.g., SEQ ID NO:12), but vary in some other manner (e.g., a post-translationalmodification). Such a polypeptide can comprise, for example, at least70%, at least 80%, at least 90% or at least 95% sequence identity to SEQID NO: 12, or have, for example, a maximum of 5, 10, 15, 20, 25, 30, 35,40, 45 or 50 amino acid differences (e.g., substitutions, deletionsand/or additions) from the amino acid sequence of SEQ ID NO: 12. In someembodiments, the PIP homolog, variant, fragment or modified form thereofretains at least 90% sequence identity or have a maximum of 25 aminoacid differences from SEQ ID NO:12. A PIP homolog, variant, fragment ormodified form retains at least one activity of human PIP.

By “FGF23” or “Fibroblast Growth Factor 23” is meant the polypeptidealso known as FGF-23, ADHR; FGFN; HPDR2; HYPF; PHPTC; External IDs:OMIM: 605380 MGI: 1891427 HomoloGene: 10771 GeneCards: FGF23 Gene;Species: Human; Entrez 8074; Ensembl ENSG00000118972; UniProt: Q9GZV9;RefSeq (mRNA): NM_020638; RefSeq (protein): NP_065689; Location (UCSC):Chr 12: 4.48-4.49 Mb; Species: Mouse; Entrez: 64654; Ensembl:ENSMUSG00000000182; UniProt: Q9EPC2; RefSeq (mRNA): NM_022657; RefSeq(protein): NP_073148; Location (UCSC): Chr 6: 127.07-127.08 Mb. TheFGF23 gene encodes the FGF23 polypeptide.

By “FGF23 peptide” as used herein is meant human FGF23 or a homolog,variant, fragment or modified form thereof, which retains at least oneactivity of human FGF23.

The sequence of a non-limiting example of human FGF23, including thesignal peptide, is presented in SEQ ID NO: 9:

(SEQ ID NO: 9)         10         20         30         40MLGARLRLWV CALCSVCSMS VLRAYPNASP LLGSSWGGLI        50         60         70         80HLYTATARNS YHLQIHKNGH VDGAPHQTIY SALMIRSEDA        90        100        110        120GFVVITGVMS RRYLCMDFRG NIFGSHYFDP ENCRFQHQTL       130        140        150        160ENGYDVYHSP QYHFLVSLGR AKRAFLPGMN PPPYSQFLSR       170        180        190        200RNEIPLIHFN TPIPRRHTRS AEDDSERDPL NVLKPRARMT       210        220        230        240PAPASCSQEL PSAEDNSPMA SDPLGVVRGG RVNTHAGGTG        250        260PEGCRPFAKF I

SEQ ID NO: 9 represents a full-length, human wild-type FGF23, includingthe signal peptide (amino acids 1-24), which is not required forfunction. Yamashita et al. 2000 Biochem. Biophys. Res. Comm. 277:494-498; Shimada et al. 2001 Proc. Natl. Acad. Sci. USA 98: 6500-6505;and Zhang et al. 2004 Protein Sci. 13: 2819-2824.

A non-limiting example of the term “FGF23” as used herein is amino acids(aa) 25-251 of SEQ ID NO: 9, which thus lacks the signal peptide (aminoacids 1-24) and is presented below as SEQ ID NO: 8.

(SEQ ID NO: 8)          10        20         30         40         50                          YPNASP LLGSSWGGLI HLYTATARNS        60         70         80         90        100YHLQIHKNGH VDGAPHQTIY SALMIRSEDA GFVVITGVMS RRYLCMDFRG       110        120        130        140        150NIFGSHYFDP ENCRFQHQTL ENGYDVYHSP QYHFLVSLGR AKRAFLPGMN       160        170        180        190        200PPPYSQFLSR RNEIPLIHFN TPIPRRHTRS AEDDSERDPL NVLKPRARMT       210        220        230        240        250PAPASCSQEL PSAEDNSPMA SDPLGVVRGG RVNTHAGGTG PEGCRPFAKF        260 IBy a “homolog,” “variant”, “fragment” or “modified form” of FGF23 or thelike is meant a polypeptide similar but non-identical to a human FGF23(e.g., SEQ ID NO: 8), but which retains at least one activity of humanFGF23. Such a polypeptide can comprise, for example, at least 70%, atleast 80%, at least 90% or at least 95% sequence identity to SEQ ID NO:8, or have, for example, a maximum of 5, 10, 15, 20, 25, 30, 35, 40, 45or 50 amino acid differences (e.g., substitutions, deletions and/oradditions) from the amino acid sequence of SEQ ID NO: 8. In someembodiments, the FGF23 homolog, variant, fragment or modified formthereof retains at least 90% sequence identity or have a maximum of 25amino acid differences from SEQ ID NO: 8. A FGF23 homolog, variant,fragment or modified form retains at least one activity of human FGF23.Such activities (or functions) include, as non-limiting examples, thoseknown for human FGF23, including roles in binding to a FGF23 receptor,interacting with Klotho protein, cellular proliferation, and cellsignaling; and activity in various in vitro assays of FGF23 activity,including the Egr-1-luciferase assay; and an activity related to aFGF23-related disease such as an age-related condition (selected fromthe group consisting of sarcopenia, skin atrophy, muscle wasting, brainatrophy, atherosclerosis, arteriosclerosis, pulmonary emphysema,osteoporosis, osteoarthritis, immunologic incompetence, high bloodpressure, dementia, Huntington's disease, Alzheimer's disease,cataracts, age-related macular degeneration, prostate cancer, stroke,diminished life expectancy, memory loss, wrinkles, impaired kidneyfunction, and age-related hearing loss), a metabolic disorder (selectedfrom the group consisting of Type II Diabetes, Metabolic Syndrome,hyperglycemia, and obesity), hyperphosphatemia (tumoral calcinosis,hyperphosphatemic hyperostosis syndrome), chronic renal disease, chronicrenal failure, cancer, breast cancer, and/or muscle atrophy. Yamashitaet al. 2000 Biochem. Biophys. Res. Comm. 277: 494-498; Shimada et al.2001 Proc. Natl. Acad. Sci. USA 98: 6500-6505; Urakawa et al. 2006Nature 444: 770-774; Zhang et al. 2004 Protein Sci. 13: 2819-2824; WO2013/027191, WO 2011/092234 and WO 2009/095372. In some embodiments, theinvention provides a conjugate comprising a fatty acid described hereinand a FGF23 peptide, wherein the FGF23 peptide retains at least oneactivity of FGF23; and in some embodiments, the activity of FGF23retained is function in the in vitro Egr-1-luciferase assay.

By a “homolog” of FGF23 is meant a polypeptide corresponding to humanFGF23, but from a different source, such as a mammal, such as mouse,rat, cynomolgus monkey, cow, pig, sheep, horse, dog, etc., yet retainsat least one function of human FGF23.

By a “variant” of FGF23 is meant a FGF23 which comprises one or moremutation (e.g., deletion, substitution or addition), e.g., relative toSEQ ID NO: 8, yet retains at least one function of human FGF23.Mutations in FGF23 include those at positions Y154, Q156, R176, R179,C206 and C244. Such mutations have previously been described. A mutationat R179 confers proteolysis resistance on FGF23; in ADHR, mutations ofthe 176RXXR179 site prevent cleavage and inactivation of FGF23. White etal. 2000 Nat. Genet. 26: 345-348; Liu et al. 2003 J. Biol. Chem. 278:37419-37426. The mutation at Y154 decreases degradation; mutation atQ156 eliminates a cleavage site; and mutations at C206 and C244 reducedimerization and aggregation. WO 2013/027191 and WO 2011/092234. A FGF23homolog, variant, or modified form can further comprise one or moreadditional amino acids (which are not normally found in wild-type humanFGF23).

A non-limiting example of a FGF23 variant is shown here:

(SEQ ID NO: 10)          10        20         30         40         50                         MYPNASP LLGSSWGGLI HLYTATARNS        60         70         80         90        100YHLQIHKNGH VDGAPHQTIY SALMIRSEDA GFVVITGVMS RRYLCMDFRG       110        120        130        140        150NIFGSHYFDP ENCRFQHQTL ENGYDVYHSP QYHFLVSLGR AKRAFLPGMN       160        170        180        190        200PPPYSQFLSR RNEIPLIHFN TPIPRRHTQS AEDDSERDPL NVLKPRARMT       210        220        230        240        250PAPASCSQEL PSAEDNSPMA SDPLGVVRGG RVNTHAGGTG PEGCRPFAKF        260 I 

SEQ ID NO: 10 shows a variant of FGF23 in which the signal peptide (aa1-24) has been deleted, but an initial M at position 1 has beenre-introduced; and the amino acid corresponding to R179 has been mutatedto Q. SEQ ID NO: 10 is also designated “hFGF23R 179Q”, “FGF23 R179”,“hFGF23 (R179Q)” and the like and represents a FGF23 variant which isused in Example 28C.

Additional FGF23 variants include, as non-limiting examples, those whichhave the sequence of SEQ ID NO: 8 or SEQ ID NO: 10, but also have amutation at one or more of: Y154, Q156, R176, R179, C206 and C244.Additional FGF23 variants include, as non-limiting examples, those whichhave the sequence of SEQ ID NO: 8 or SEQ ID NO: 10, but also have amutation at one or more of: Y154, Q156, R176, R179, C206 and C244 andfurther comprise one or more additional amino acids (which are notnormally found in wild-type human FGF23).

By a “fragment” of FGF23 is meant a FGF23 which comprises one or moredeleted amino acids, e.g., relative to SEQ ID NO: 8, yet retains atleast one function of human FGF23. Functional fragments of FGF23 includeamino acids 180-251 of SEQ ID NO: 8, Goetz et al. 2010 Proc. Natl. Acad.Sci. USA 107: 407-412. A FGF23 fragment can also have one or mutations,e.g., at any one or more of positions Y154, Q156, R176, R179, C206 andC244, but can retain at least one activity of human FGF23.

By “modified form” of FGF23 is meant a FGF23 which comprises a sequencesimilar or identical to that of FGF23, e.g., SEQ ID NO: 8, but which hasone or more modification, and which retains at least one activity ofhuman FGF23. Such a modification can include, as non-limiting examples,a post-translational modification (phosphorylation, methylation, oraddition of a carbohydrate), or conjugation to a second moiety, which isnot FGF23. Such a second moiety can be, as non-limiting examples, asignal peptide, alpha or beta Klotho or a fragment thereof (e.g.,soluble Klotho or sKlotho), a Fc (e.g., FcLALA), or other modification.WO 2011/092234 and WO 2009/095372.

As used herein, the term “AgRP peptide or polypeptide,” and like termsrefer to the Agouti-Related Peptide, i.e., a signaling molecule made upof 132 amino acids that is post-translationally processed into itsactive or mature form, AgRP (83-132), which contains 10 cysteineresidues and forms a network of five disulfide bonds. AgRP plays a roleas an inverse agonist of the melanocortin receptors MC3R and MC4R. Theterm “AgRP peptide” in all instances includes salts thereof. In someembodiments, AgRP can be in an amide form, e.g., amidation of C-terminus—CO2H to form C(O)—NH2. In other embodiments, AgRP can be in an acidform.

The term “AgRP peptide” also includes shorter biologically activefragments of AgRP. A fragment is a portion of the parent sequence whichis identical in sequence but shorter in length than the parent sequenceand retain biological activity (i.e. inverse agonism). Fragments of AgRPpolypeptides as well as variants thereof have also been described inJackson, P. J. et al., Biochemistry 41, 7565-7572, which is incorporatedby reference herein. For example AgRP (87-120) and AgRP(87-132) possessapproximately the same MC3R and MC4R affinity as AgRP(83-132) andexhibit equivalent inverse agonism. Additional fragments of AgRPpolypeptide have been described in Christine G. Joseph et al., Peptides24 (2003), 263-270; which is incorporated by reference herein. Examplesof fragments are AgRP(86-132) and monocyclic AgRP (109-118) as well aselongation thereof at the N- and/or C-terminus.

The term “AgRP polypeptides” also encompasses “AgRP mutant polypeptide”which are AgRP polypeptide in which a naturally occurring AgRPpolypeptide sequence has been modified. Such modifications have beendescribed in PCT application No. WO2013/006656, which is incorporated byreference herein.

The terms “GDF15 peptide”, “GDF15 polypeptide” and “GDF15 protein” areused interchangeably and mean a naturally-occurring wild-typepolypeptide expressed in a mammal, such as a human or a mouse. Forpurposes of this disclosure, the term “GDF15 protein” can be usedinterchangeably to refer to any full-length GDF15 polypeptide, whichconsists of 308 amino acid residues; (NCI Ref. Seq. NP_004855.2)containing a 29 amino acid signal peptide (amino acids 1-29), a 167amino acid pro-domain (amino acids 30-196), and a mature domain of 112amino acids (amino acids 197-308) which is excised from the prodomain byfurin-like proteases. A 308-amino acid GDF15 polypeptide is referred toas “full-length” GDF15 polypeptide; a 112 amino acids GDF15 polypeptide(e.g. amino acids 197-308) is a “mature” GDF15 polypeptide. The matureGDF15 peptide contains the seven conserved cysteine residues requiredfor the formation of the cysteine knot motif (having three intrachaindisulfide bonds) and the single interchain disulfide bond that aretypical for TGF˜ superfamily members. The mature GDF15 peptide containstwo additional cysteine residues that form a fourth intrachain disulfidebond. Therefore, biologically active GDF15 is a homodimer of the maturepeptide covalently linked by one interchain disulfide bond. A GDF15protein or polypeptide therefore also includes multimer, moreparticularly dimer of the protein. Each monomeric unit which constitutethe homodimer GDF15 may be linked to a fatty acid of Formulae A1, A2 orA3.

By “GDF15” or “GDF15 protein” as used herein is also meant human GDF15or a homolog, variant, mutant, fragment or modified form thereof, whichretains at least one activity of human GDF15.

The term “GDF15 mutant polypeptide” or “GDF15 variant polypeptide”encompasses a GDF15 polypeptide in which a naturally occurring GDF15polypeptide sequence has been modified. Such modifications include, butare not limited to, one or more amino acid substitutions, includingsubstitutions with non-naturally occurring amino acidsnon-naturally-occurring amino acid analogs and amino acid mimetics.

In one aspect, the term “GDF15 mutant protein” or “GDF15 variantpolypeptide” refers to a GDF15 protein sequence in which at least oneresidue normally found at a given position of a native GDF15 polypeptideis deleted or is replaced by a residue not normally found at thatposition in the native GDF15 sequence. In some cases it will bedesirable to replace a single residue normally found at a given positionof a native GDF15 polypeptide with more than one residue that is notnormally found at the position; in still other cases it may be desirableto maintain the native GDF15 polypeptide sequence and insert one or moreresidues at a given position in the protein; in still other cases it maybe desirable to delete a given residue entirely; all of these constructsare encompassed by the term “GDF 15 mutant protein” or “GDF15 variantprotein”. In one aspect of the invention, the GDF15 mutant protein or“GDF15 variant protein” has a sequence selected from any one of SEQ IDNO 1 to SEQ ID No 7. The present invention also encompasses nucleic acidmolecules encoding such GDF15 mutant polypeptide sequences or GDF15variant polypeptide sequences.

By a “homolog,” “variant”, “fragment” or “modified form” of GDF15 or thelike is meant a polypeptide similar but non-identical to a human GDF15,but which retains at least one activity of human GDF15.

By “modified form” of GDF15 is meant a GDF15 which comprises a sequencesimilar or identical to that of GDF15, but which has one or moremodification, and which retains at least one activity of human GDF15.Such a modification can include, as non-limiting examples, apost-translational modification (phosphorylation, methylation, oraddition of a carbohydrate).

By a “homolog” of GDF15 is meant a polypeptide corresponding to humanGDF15, but from a different source, such as a mammal, such ascynomolgous monkeys, mice and rats etc., yet retains at least onefunction of human GDF15. In some instances, a GDF15 homolog can be usedto treat or ameliorate a metabolic disorder in a subject in a matureform of a GDF15 mutant polypeptide that is derived from the same speciesas the subject.

In various embodiments, a GDF15 polypeptide, homolog, variant, mutant,fragment or modified form thereof comprises an amino acid sequence thatis at least about 85 percent identical to a naturally-occurring GDF15protein. In other embodiments, a GDF15 polypeptide comprises an aminoacid sequence that is at least about 90 percent, or about 95, 96, 97,98, or 99 percent identical to a naturally-occurring GDF15 polypeptideamino acid sequence. Such GDF15 polypeptide, homolog, variant, mutant,fragment or modified form thereof possess at least one activity of awild-type GDF15 mutant polypeptide, such as the ability to lower bloodglucose, insulin, triglyceride, or cholesterol levels; the ability toreduce body weight; or the ability to improve glucose tolerance, energyexpenditure, or insulin sensitivity.

In various respective embodiments, a GDF15 polypeptide or homolog,variant, mutant, fragment or modified form thereof has a biologicalactivity that is equivalent to, greater to or less than that of thenaturally occurring form of the mature GDF15 protein. Examples ofbiological activities include the ability to lower blood glucose,insulin, triglyceride, or cholesterol levels; the ability to reduce bodyweight; or the ability to improve glucose tolerance, lipid tolerance, orinsulin sensitivity; the ability to lower urine glucose and proteinexcretion.

As used herein in the context of the structure of a polypeptide orprotein, the term “N-terminus” (or “amino terminus”) and “C-terminus”(or “carboxyl terminus”) refer to the extreme amino and carboxyl ends ofthe polypeptide, respectively.

The term “therapeutic polypeptide” or “therapeutic protein” as usedherein means a polypeptide or protein which is being developed fortherapeutic use, or which has been developed for therapeutic use.

The linker separates the biomolecule and the fatty acid moiety. Itschemical structure is not critical, since it serves primarily as aspacer.

The linker is a chemical moiety that contains two reactivegroups/functional groups, one of which can react with the biomoleculeand the other with the fatty acid moiety. The two reactive/functionalgroups of the linker are linked via a linking moiety or spacer,structure of which is not critical as long as it does not interfere withthe coupling of the linker to the biomolecule and the fatty acid moietyof Formula A1, A2 or A3.

The linker can be made up of amino acids linked together by peptidebonds. In some embodiments of the present invention, the linker is madeup of from 1 to 20 amino acids linked by peptide bonds, wherein theamino acids are selected from the 20 naturally occurring amino acids. Invarious embodiments, the 1 to 20 amino acids are selected from the aminoacids glycine, serine, alanine, methionine, asparagine, glutamine,cysteine and lysine. In some embodiments, a linker is made up of amajority of amino acids that are sterically unhindered, such as glycineand alanine. In some embodiments, linkers are polyglycines,polyalanines, combinations of glycine and alanine (such aspoly(Gly-Ala)), or combinations of glycine and serine (such aspoly(Gly-Ser)). In some embodiments, a linker is made up of a majorityof amino acids selected from histidine, alanine, methionine, glutamine,asparagine and glycine. In some embodiments, linkers containpoly-histidine moiety.

In some embodiments, the linker comprises 1 to 20 amino acids which areselected from unnatural amino acids. While a linker of 1-10 amino acidresidues is preferred for conjugation with the fatty acid moiety, thepresent invention contemplates linkers of any length or composition. Anexample of non-natural amino acid linker is 8-Amino-3,6-dioxaoctanoicacid having the following formula:

or its repeating units.

The linkers described herein are exemplary, and linkers that are muchlonger and which include other residues are contemplated by the presentinvention. Non-peptide linkers are also contemplated by the presentinvention.

In other embodiments, the linker comprise one or more alkyl groups,alkenyl groups, cycloalkyl groups, aryl groups, heteroaryl groups,heterocyclic groups, polyethylene glycol and/or one or more natural orunnatural amino acids, or combination thereof, wherein each of thealkyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, polyethyleneglycol and/or the natural or unnatural amino acids are optionallycombined and linked together, or linked to the biomolecule and/or to thefatty acid moiety, via a chemical group selected from —C(O)O—, —OC(O)—,—NHC(O)—, —C(O)NH—, —O—, —NH—, —S—, —C(O)—, —OC(O)NH—, —NHC(O)—O—,═NH—O—, ═NH—NH— or ═NH—N(alkyl)-.

Linkers containing alkyl spacer are for example —NH—(CH₂)_(z)—C(O)— or—S—(CH₂)_(z)—C(O)— or —O—(CH₂)_(z)—C(O)—, —NH—(CH₂)_(z)—NH—,—O—C(O)—(CH₂)_(z)—C(O)—O—, —C(O)—(CH₂)_(z)—O—,—NHC(O)—(CH₂)_(z)—C(O)—NH— and the like wherein z is 2-20 can be used.These alkyl linkers can further be substituted by any non-stericallyhindering group, including, but not limited to, a lower alkyl (e.g.,C₁-C₆), lower acyl, halogen (e.g., Cl, Br), CN, NH₂, or phenyl.

The linker can also be of polymeric nature. The linker may includepolymer chains or units that are biostable or biodegradable. Polymerswith repeat linkage may have varying degrees of stability underphysiological conditions depending on bond lability. Polymers maycontain bonds such as polycarbonates (—O—C(O)—O—), polyesters(—C(O)—O—), polyurethanes (—NH—C(O)—O—), polyamide (—C(O)—NH—). Thesebonds are provided by way of examples, and are not intended to limit thetype of bonds employable in the polymer chains or linkers of theinvention. Suitable polymers include, for example, polyethylene glycol(PEG), polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids,divinylether maleic anhydride, N-(2-hydroxypropyl)-methacrylicamide,dextran, dextran derivatives, polypropylene glycol, polyoxyethylatedpolyol, heparin, heparin fragments, polysaccharides, cellulose andcellulose derivatives, starch and starch derivatives, polyalkyleneglycol and derivatives thereof, copolymers of polyalkylene glycols andderivatives thereof, polyvinyl ethyl ether, and the like and mixturesthereof. A polymer linker is for example polyethylene glycol (PEG). ThePEG linker can be linear or branched. A molecular weight of the PEGlinker in the present invention is not restricted to any particularsize, but certain embodiments have a molecular weight between 100 to5000 Dalton for example 500 to 1500 Dalton.

The linker contains appropriate functional-reactive groups at bothterminals that form a bridge between the amino group of the peptide orpolypeptide/protein and a functional/reactive group on the fatty acidmoiety (e.g the carboxylic acid functionality of the fatty acid moietyof formula A1, A2 and A3).

The linker may comprise several linking moieties (or spacer) ofdifferent nature (for example a combination of amino acids, heterocyclylmoiety, PEG and/or alkyl moieties). In this instance, each linkingmoiety contains appropriate functional-reactive groups at both terminalsthat form a bridge between the amino group of the peptide orpolypeptide/protein and the next linking moiety of different natureand/or contains appropriate functional-reactive groups that form abridge between the prior linking moiety of different nature and thefatty acid moiety.

The modified peptides or polypeptides and/or peptide-polypeptide partialconstruct (i.e. peptide/polypeptide attached to a partial linker)include reactive groups which can react with available reactivefunctionalities on the fatty acid moiety (or modified fatty acid moiety:i.e. already attached a partial linker) to form a covalent bond.Reactive groups are chemical groups capable of forming a covalent bond.Reactive groups are located at one site of conjugation and can generallybe carboxy, phosphoryl, acyl group, ester or mixed anhydride, maleimide,N-hydroxysuccinimide, tetrazine, alkyne, imidate,pyridine-2-yl-disulfanyl, thereby capable of forming a covalent bondwith functionalities like amino group, hydroxyl group, alkene group,hydrazine group, hydroxylamine group, an azide group or a thiol group atthe other site of conjugation.

Reactive groups of particular interest for conjugating a biomolecule ormodified biomolecule to a linker and/or a linker to the fatty acidmoiety and/or to conjugate various linking moieties of different naturetogether are N-hydroxysuccinimide, alkyne (more particularlycyclooctyne).

Functionalities include: 1. thiol groups for reacting with maleimides,tosyl sulfone or pyridine-2-yldisulfanyl; 2. amino groups (for exampleamino functionality of an amino acid) for bonding to carboxylic acid oractivated carboxylic acid (e.g. amide bond formation viaN-hydroxysuccinamide chemistry), phosphoryl groups, acyl group or mixedanhydride; 3. Azide to undergo a Huisgen cycloaddition with a terminalalkyne and more particularly cyclooctyne (more commonly known as clickchemistry); 4. carbonyl group to react with hydroxylamine or hydrazineto form oxime or hydrazine respectively; 5. Alkene and more particularlystrained alkene to react with tetrazine in an aza [4+2] addition. Whileseveral examples of linkers and functionalities/reactive group aredescribed herein, the invention contemplates linkers or any length andcomposition.

EMBODIMENTS

Various embodiments of the invention are described herein. It will berecognized that features specified in each embodiment may be combinedwith other specified features to provide further embodiments.

In embodiment 1, the invention pertains to a conjugate comprising abiomolecule linked to an fatty acid moiety via a linker wherein thefatty acid moiety has the following Formulae A1, A2 or A3:

R¹ is CO₂H, H;R², R³ and R⁴ are independently of each other H, OH, CO₂H, —CH═CH₂ or—C═CH;Ak is a branched C₆-C₃₀alkylene;n, m and p are independently of each other an integer between 6 and 30;or an amide, an ester or a pharmaceutically acceptable salt thereof.

In a further aspect of embodiment 1, the conjugate according toembodiment 1 may further comprise a fatty acid of Formula A1, A2 or A3as described supra. In view of the difficulties of achieving selectiveconjugation and/or achieving mono conjugation of a fatty acid to abiomolecule, the conjugates of the invention, may comprise a biomoleculewhich is linked to one or more fatty acid moieties of Formula A1, A2 orA3. Additionally, in view of the multimeric nature of some proteins,each monomeric unit which constitutes a multimeric protein, may belinked to a fatty acid moiety, but not all monomeric units have tonecessarily be linked to a fatty acid moiety as long as at least one ofthe monomeric unit is linked to a fatty acid moiety. In a furtheraspect, the invention relates to mixtures of the conjugates of theinvention. For example, the mixture may comprise a biomolecule, forexample a multimeric biomolecule, for example a dimeric biomolecule,which is linked to one fatty acid moietie of Formula A1, A2 or A3, and abiomolecule, for example a multimeric biomolecule, for example a dimericbiomolecule, which is linked to more than one fatty acid moieties ofFormula A1, A2 or A3. Examples of the invention below further highlightthis aspect of selective or non-selective multiconjugation of fattyacids to a protein or polypeptide.

In embodiment 1A, the invention pertains to a conjugate according toembodiment 1 wherein the fatty acid moiety is of Formula A1. In aparticular aspect of this embodiment, the conjugate comprises a fattyacid moiety of Formula A1 wherein n and m are independently 8 to 20,preferably 10 to 16. In another aspect of this embodiment, the inventionpertains to a conjugate according to embodiment 1 or 1A wherein thefatty acid moiety is of Formula A1 and wherein at least one of R² and R³is CO₂H.

In embodiment 2, the invention pertains to a conjugate according toembodiment 1 or 1A, wherein the fatty acid moiety is selected from thefollowing Formulae:

wherein Ak³, Ak⁴, Ak⁵, Ak⁶ and Ak⁷ are independently a (C₈₋₂₀)alkylene,R⁵ and R⁶ are independently (C₈₋₂₀)alkyl.

In embodiment 3, the invention pertains to a conjugate according toembodiment 1, 1A or 2 wherein the fatty acid moiety is selected from thefollowing Formulae:

In embodiment 3A, the invention pertains to a conjugate according toembodiment 1, 1A or 2 wherein the fatty acid moiety is selected from thefollowing Formulae:

In embodiment 3B, the invention pertains to a conjugate according toembodiment 1 wherein the fatty acid moiety is of Formula A2 or A3. In aparticular aspect of this embodiment, the conjugate comprises an fattyacid moeity of Formula A2 wherein p is 8 to 20, or a fatty acid moeityof Formula A3 wherein Ak is C₈₋₂₀alkylene.

In embodiment 3C, the invention pertains to a conjugate according toembodiment 1 or 3B wherein the fatty acid moeity is selected from thefollowing Formulae:

wherein Ak² is C₈₋₂₀alkylene.

In embodiment 4, the invention pertains to a conjugate according to anyof the preceeding embodiments wherein the linker comprise one or morealkyl groups, alkenyl groups, cycloalkyl groups, aryl groups, heteroarylgroups, heterocyclic groups, polyethylene glycol, one or more natural orunnatural amino acids, or combination thereof, wherein each of thealkyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, polyethyleneglycol and/or the natural or unnatural amino acids are optionallycombined and linked together or linked to the biomolecule and/or to thefatty acid moiety via a chemical group selected from —C(O)O—, —OC(O)—,—NHC(O)—, —C(O)NH—, —O—, —NH—, —S—, —C(O)—, —OC(O)NH—, —NHC(O)—O—,═NH—O—, ═NH—NH— or ═NH—N(alkyl)-.

In embodiment 5, the invention pertains to a conjugate according to anyof the preceeding embodiment, wherein the linker comprises an unbranchedoligo ethylene glycol moiety of Formula:

wherein y is 0 to 34.

In embodiment 6, the invention pertains to conjugate according to any ofthe preceeding embodiments wherein the linker comprises (or furthercomprises) a heterocyclic moiety selected from the following Formulae:

Such heterocyclyl containing linkers are obtained for example byazide-alkyne Huisgen cycloaddition, which more commonly known as clickchemistry. More particularly, some of the heterocyclyl depicted supraresult from the reaction of a cycloalkyne with an azide-containingmoiety.

Cycloalkyne are readily available from commercial sources and cantherefore be functionalized via cycloaddition with a moiety containingan azide functionality (e.g. a linker containing a terminal azidefunctionality). Examples of the use of cyclic alkyne click chemistry inprotein labeling has been described in US 2009/0068738 which is hereinincorporated by reference.

Non-limiting examples of cycloakyne agents which can be used in Huisgencycloaddition are:

In embodiment 6A, the invention pertains to a conjugate according to anyone of embodiments 1 to 5, wherein the linker comprises (or furthercomprises) a heterocyclyl selected from the following Formulae:

wherein r is an integer of 0 to 2 and s is an integer of 0 to 3.

Such heterocyclic linkers can be obtained via an aza [4+2] cycloadditonof an alkene, or preferably a strained alkene such as cycloalkane, withthe following moiety:

wherein Rf is for example —CH₂NH₂, —OH, —CH₂—CO₂H, —S—CH₂—CO₂H,—(O—CH₂)₄₋₆—C(O)—OH— or

Such tetrazine moieties are readily available from commercial sourcesand can react with an alkene-containing moiety, for example a linkercontaining terminal alkene functionality.

In embodiment 6B, the invention pertains to a conjugate according to anyone of embodiments 1 to 5 wherein the linker comprises (or furthercomprises) a heterocyclyl of Formula:

Such heterocyclic moiety can be obtained by reacting a maleimide with athiol containing moiety, such as for example a linker containing aterminal thiol functionality.

These reagents which are readily available and/or commercially availableare attached directly or via a linker as described supra to the peptideor polypeptide of interest. The alkyne, maleimide or tetrazine reactivegroups are reacted with a functional group (azide, thiol and alkenerespectively) which is present on the fatty acid moiety or on alinker-fatty acid construct (such as for example a PEG-fatty acidconstruct).

In embodiment 7, the invention pertains to a conjugate according to anyof the preceeding embodiments wherein the linker comprises or furthercomprises one or more amino acids independently selected from histidine,methionine, alanine, glutamine, asparagine and glycine. In oneparticular aspect of this embodiment, the linker comprises 1 to 6 aminoacid selected from histidine, alanine and methionine.

In embodiment 8, the invention pertains to a conjugate according to anyone of the preceeding embodiments wherein the biomolecule is a peptideor polypeptide. In one particular aspect of embodiment 8, the inventionpertains to a conjugate according to any one of the preceedingembodiments wherein the peptide or polypeptide is 1) human GrowthDifferentiation Factor 15 (GDF15), homologs, variants, mutants,fragments and other modified forms thereof; 2) an APJ agonist peptide,3) an oxytocin receptor agonist peptide, 4) serelaxin, 5) NPFF, 6) a PIPpeptide, 7) an FGF23 peptide 8) an AgRP peptide or 9) a siRNA.

In embodiment 8A, the invention pertains to a conjugate according to anyone of the preceeding embodiments wherein the biomolecule is humanGrowth Differentiation Factor 15 (GDF15), homologs, variants, mutants,fragments and other modified forms thereof; or a dimer thereof. In oneaspect of this embodiment, the biomolecule is human GrowthDifferentiation Factor 15 (GDF15) mutant or variant. In a preferredembodiment, the biomolecule is a dimer of GDF15 or a variant or mutantthereof. In view of the homodimer nature of the GDF15 polypeptide ormutant or variant thereof, each of the two polypeptide chains (i.e. eachmonomeric unit) which constitute the homodimer, may be linked to a fattyacid molecule of Formula A1, A2 or A3 via a linker. Therefore the GDF15homodimer may be linked to one or two fatty acids via a linker. Thestructure of the GDF15 linked to a fatty acid moiety via a linker may berepresented as follow:

wherein FA represent the fatty acid moiety and L the linker, and GDF15monomer unit 1 and monomer unit 2 are both linked to a fatty acid moietyvia a linker; or

wherein FA is the fatty acid moiety and L the linker and only one of themonomer unit is linked to a fatty acid moiety via the linker and whereinthe line between the 2 monomeric units represent a disulfide bond.Furthermore, the invention also relates to a mixture comprising aconjugate of structure A and a conjugate of structure B.

In embodiment 8B, the invention contemplates a conjugate according toembodiment 8A wherein the human GDF15 mutant is obtained by replacementof one or more amino acid residues of the mature polypeptide withanother residue. In one particular aspect of this embodiment, the lasttwo amino acid residues at the N-terminal of human GDF15 (i.e Arginine198 and Alanine 197) have been replaced with an amino acid sequence XH—wherein H is histidine and X is an amino acid selected from methionine,alanine, glutamine, asparagine and glycine. In a preferred aspect ofthis embodiment, the hGDF15 mutant is MH(199-308)hGDF15 orAH(199-308)hGDF15 (SEQ ID. NO:7).

In embodiment 8C, the last three amino acid residues at the N-terminalof human GDF15 (i.e. Asparagine 199, Arginine 198 and Alanine 197) havebeen replaced with an amino acid sequence XHX′— wherein H is histidineand X′ and X are amino acids independently selected from selected frommethionine, alanine, glutamine, asparagine and glycine. In anotheraspect of this embodiment, the last three amino acid residues at theN-terminal of human GDF15 (i.e. Asparagine 199, Arginine 198 and Alanine197) have been replaced with an amino acid sequence AHX′— wherein H ishistidine and X′ is an amino acids independently selected from selectedfrom methionine, alanine, glutamine, asparagine and glycine. In apreferred aspect of this embodiment, the modified GDF15 protein isMHA(200-308)hGDF15 or AHA(200-308)hGDF15.

Compared to the native GDF15 protein, the GDF15 mutant enables theselective labeling of the protein at the N-terminus (i.e. conjugation ofthe fatty acid at the preferred N-terminus of the GDF15). The selectivelabeling of peptide and protein is described in further details inco-filed US application Nos. 62/015,858 and 62/082,337.

In embodiment 8D, the invention pertains to a conjugate according to anyone of embodiments 1 to 8 wherein the biomolecule is an APJ agonistpeptide. In a particular aspect of this embodiment, the APJ agonistpeptide is a peptide described in patent PCT application numbers WO2013/111110, WO 2014/081702, WO 2015/013168, WO 2015/013165, WO2015/013167 and WO 2015/013169 which are herein incorporated byreference.

In embodiment 8E, the invention pertains to a conjugate according to anyone of embodiments 1 to 8 wherein the biomolecule is an oxytocinreceptor agonist peptide. In a particular aspect of this embodiment, theoxytocin receptor agonist peptide is a peptide described in patent PCTapplication numbers WO 2009/122285 (Ferring B. V.) and WO 2014/095773(Hoffman-La Roche) which are herein incorporated by reference.

In embodiment 8F, the invention pertains to a conjugate according to anyone of embodiments 1 to 8 wherein the biomolecule is an AgRP peptide. Ina particular aspect of this embodiment, the AgRP peptide is AgRP(83-132)wherein the C-terminus is in the form of a—free CO₂H or an amide thereof(e.g. —C(O)NH₂).

In embodiment 8G, the invention pertains to a conjugate according to anyone of embodiments 1 to 8 wherein the biomolecule is an FGF23 peptide.In a particular aspect of this embodiment, the FGF23 peptide is a FGF23variant of SEQ ID NO: 8 having a mutation at R179 and optionally one ormore additional mutations at Y154, Q156, R176, R179, C206 and C244.

In another particular aspect of this embodiment, the FGF23 peptide is aFGF23 variant of SEQ ID NO: 8 having mutations at R179, Q156, C206 andC244.

In embodiment 8H, the invention pertains to a conjugate according to anyone of embodiments 1 to 8 wherein the biomolecule is Serelaxin.

In embodiment 8I, the invention pertains to a conjugate according to anyone of embodiments 1 to 8 wherein the biomolecule is NPFF peptide.

In embodiment 8J, the invention pertains to a conjugate according to anyone of embodiments 1 to 8 wherein the biomolecule is a PIP peptide. In aparticular aspect of this embodiment, the PIP peptide is the histaggedprotein MHHHHHHH-PIP wherein PIP is of SEQ ID NO: 12.

In embodiment 8K, the invention pertains to a conjugate according to anyone of embodiments 1 to 8 wherein the biomolecule is a siRNA.

In embodiment 8L, the invention pertains to a conjugate according to anyone of the proceeding embodiments, further comprising a second fattyacid moiety linked to the biomolecule via a linker. Preferably the twofatty acid-linker moieties are of the same structure.

In embodiment 9, the invention pertains to a conjugate according toembodiment 1, 2, 8, 8A, 8B or 8C having the following structure:

wherein hGDF15* is hGDF15 wherein the 2 or 3 amino acid at theN-terminus have been replaced with an amino acid sequence XH— or XHX′—respectively,wherein H is histidine and X and X′ are independently selected from Mand A; or a dimer thereof; andwherein his-hGDF15 is hGDF15 wherein a tag, comprising 1 to 6 histidineamino acids and optionally 1 or 2 methionine amino acids, has been addedto the N-terminus of hGDF15; or a dimer thereof; ands is an integer between 20-30

In one aspect of this embodiment the tag comprises histidine amino acidsand 1 or 2 non-adjacent methionine amino acids. In another aspect ofthis embodiment, the arrangement of histidine and methionine amino acidsis so that the amino acid at the position adjacent to the N-terminusamino acid is a histidine. In a further aspect of this embodiment thetag is selected from MHHHHHHM- (SEQ ID. NO: 15) and MHHHHHH- (SEQ ID.NO: 16).

In a particular aspect of embodiment 9, in view of the homodimer natureof hGDF15* and his-hGDF15, one or two polypeptide chains (monomericunit) which constitute the homodimer may be linked the fatty acidmolecule via a linker. As a result, the homodimer may be linked to oneor may be linked to two fatty acid molecules via a linker at theN-terminus. Such embodiment may be represented by the GDF15 biomoleculelinked to the fatty acid via a linker having the Formulae below:

wherein both monomeric units of his-hGDF15 or of hGDF15* (as definedabove) are linked to the fatty acid moiety via a linker at bothN-terminus; or

wherein only one of the monomer unit of his-hGDF15 or of hGDF15* (asdefined above) is linked to the fatty acid moiety via a linker at theN-terminus. Furthermore, the invention also contemplates mixtures ofconjugates of the invention; for example a mixture comprising aconjugate of Formula C and a conjugate of Formula E, or a mixturecomprising a conjugate of formula D and a conjugate of Formula F.

In embodiment 10, the invention pertains to a composition comprising amixture of a conjugate of Formula C and a conjugate of Formula E. Inembodiment 10A, the invention pertains to a composition comprising amixture of a conjugate of Formula D and a conjugate of Formula F.

Therefore, in embodiment 10B, the invention relates to a conjugateaccording to claim 1, 2, 9 or 10, comprising:

-   -   1. a variant of homodimer hGDF15 wherein the 2 or 3 amino acid        at the N-terminus have been replaced with an amino acid sequence        XH— or XHX′— respectively, wherein H is histidine and X and X′        are independently selected from M and A; or        -   a homodimer hGDF15 wherein a tag, comprising 1 to 6            histidine amino acids and optionally 1 or 2 methionine amino            acids, has been added at the N-terminus of hGDF15; and    -   2. one or two fatty acid of Formula:

wherein the fatty acid is linked to the N-terminus of the polypeptidechain via a linker; or a mixture of conjugates.

In embodiment 10C, the invention pertains to a conjugate of embodiment9, 10, 10A or 10B, wherein hGDF15* is hGDF15 wherein the 2 or 3 aminoacid at the N-terminus have been replaced with an amino acid sequenceXH— or XHX′— respectively,

wherein H is histidine and X and X′ are independently selected from Mand A; or a dimer thereof; and

wherein his-hGDF15 is hGDF15 wherein a tag, comprising 4 to 6 histidineamino acids and 1 or 2 methionine amino acids, has been added to theN-terminus of hGDF15; or a dimer thereof;

and s is an integer between 22 and 28. In one aspect of this embodimentthe tag comprises histidine amino acids and 1 or 2 non-adjacentmethionine amino acids. In another aspect of this embodiment, thearrangement of histidine and methionine amino acids is so that the aminoacid at the position adjacent to the N-terminus amino acid is ahistidine. In a further aspect of this embodiment the tag is selectedfrom MHHHHHHM- (SEQ ID. NO: 15) and MHHHHHH- (SEQ ID. NO: 16).

In embodiment 11, the invention pertains to a conjugate according to anyone of the preceeding embodiments wherein the biomolecule is selectedfrom M-(His)6-hGDF15 (SEQ ID No 1), M-(his)6-M-hGDF15 (SEQ ID NO: 2),MH(199-308)hGDF15 (SEQ ID NO: 4), MHA(200-308)hGDF15 (SEQ ID NO: 6),AHA(200-308)hGDF15 (SEQ ID NO: 7) and AH(199-308)GDF15 (SEQ ID NO: 5);or a dimer thereof.

In embodiment 11A, the invention pertains to a conjugate according toembodiment 11 wherein the biomolecule is selected from MH(199-308)hGDF15(SEQ ID NO: 4), MHA(200-308)hGDF15 (SEQ ID NO: 6), AHA(200-308)hGDF15(SEQ ID NO: 7) and AH(199-308)GDF15 (SEQ ID NO: 5); or a dimer thereof.

In embodiment 11B, the invention pertains to a conjugate according toembodiment 11 wherein the biomolecule is selected AHA(200-308)hGDF15(SEQ ID NO: 7); or a dimer thereof.

In embodiment 12, the invention pertains to a conjugate according toembodiment 11B wherein the biomolecule linked to the fatty acid via alinker is of Formula G or of Formula H:

wherein AHA-hGDF15 is SEQ ID NO: 7 and the fatty acid is linked at theN-terminus of one or of the 2 monomeric units. Furthermore, theinvention contemplates a mixture comprising the conjugate of Formula Gand the conjugate of Formula H.

In embodiment 13, the invention pertains to a composition comprising amixture of a conjugate according to embodiment 12 having Formula G and aconjugate according to embodiment 12 having Formula H. In a particularaspect of this embodiment, the mixture is a 1:1 molar ratio of aconjugate of Formulae G and a conjugate of Formula H.

The AHA-(200-308)-hGDF15 (SEQ ID NO: 7) was designed to remove theclipping site observed within the native protein as well as to removethe potential methioinine (M1) formylation site and the N-199deamidation site. The superior quality and homogeneity of the AHA wasconfirmed by a material quality check showing no clipping, deamidation,or methionine oxidation which was observed with the hGDF15 nativesequence.

MHHHHHH-ARN-(200-308)- AHA-(200-308)-hGDF15 hGDF15 (SEQ ID. NO: 17) (SEQID. NO: 7) clipping R9/N10 (<1%) None detected N10/G11 (<1%) MethionineM1: 12.0% oxidation N/A oxidation N-199 N10: 50.1% deamidation N/Adeamidation

In embodiment 14, the invention pertains to a conjugate according to anyone of preceeding embodiments wherein the fatty acid residue is attachedthe N-terminus of the peptide or protein via a linker. In embodiment 15,the invention pertains to a conjugate according to any one of thepreceeding embodiments wherein the conjugate has a plasma stabilityhalf-life of more than 5 h. In one aspect of this embodiment, theconjugate has a plasma stability half-life of more than 10 h. In anotheraspect of this embodiment, the conjugate has a plasma stabilityhalf-life of more than 20 h or more than 30 h. In yet another aspect ofthis embodiment, the conjugate has a plasma stability half-life of morethan 40 h or more than 50 h.

In embodiment 16, the invention pertains to a conjugate according to anyone of the preceeding embodiments where the improvement of plasmastability half-life compared to the non-conjugated biomolecule is 2fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold or 75 fold.

In another embodiment, the biomolecule, the linker and the fatty acidmoiety (R¹ to R⁴, n, m p and Ak) are those defined by in the Examplessection below.

In one embodiment, the invention pertains to a compound of Formula:

R¹ is CO₂H or H;R² and R³ are independently of each other H, OH, CO₂H, —CH═CH₂ or —C═CH;with the proviso that R² and R³ are not identical;R⁴ is CO₂H;n and m are independently of each other an integer between 6 and 30; oran amide, ester or pharmaceutically acceptable salt thereof. In anotheraspect of this embodiment, the invention pertains to a compound ofFormula A1 wherein at least one of R² and R³ is CO₂H. In yet a furtheraspect of this embodiment, the invention pertains to a compound selectedfrom the group consisting of:

Synthesis of Peptide/Polypeptide and/or Modified Form Thereof

The peptides or polypeptides of the invention may be produced by eithersynthetic chemical processes or by recombinant methods or combination ofboth methods. The peptides or polypeptides/protein constructs may beprepared as full-length or may be synthesized as non-full lengthfragments and joined. The peptides and polypeptides of the presentinvention can be produced by the per se known procedures for peptidesynthesis. The methods for peptide synthesis may be any of a solid-phasesynthesis and a liquid-phase synthesis. Thus, the peptide andpolypeptide of interest can be produced by condensing a partial peptideor amino acid capable of constituting the protein with the residual partthereof and, when the product has a protective group, the protectivegroup is detached whereupon a desired peptide can be manufactured. Theknown methods for condensation and deprotection include the proceduresdescribed in the following literature (1)-(5).

-   -   (1) M. Bodanszky and M. A. Ondetti, Peptide Synthesis,        Interscience Publishers, New York, 1966,    -   (2) Schroeder and Luebke, The Peptide, Academic Press, New York,        1965,    -   (3) Nobuo Izumiya et al. Fundamentals and Experiments in Peptide        Synthesis, Maruzen, 1975,    -   (4) Haruaki Yajima and Shumpei Sakakibara, Biochemical        Experiment Series 1, Protein Chemistry IV, 205, 1977, and    -   (5) Haruaki Yajima (ed.), Development of Drugs-Continued, 14,        Peptide Synthesis, Hirokawa Shoten.

After the reaction, the peptide or polypeptide can be purified andisolated by a combination of conventional purification techniques suchas solvent extraction, column chromatography, liquid chromatography,size exclusion chromatography and ion exchange chromatography andrecrystallization. Where the peptide isolated as above is a freecompound, it can be converted to a suitable salt by the known method.Conversely where the isolated product is a salt, it can be converted tothe free peptide by the known method.

The amide of polypeptide can be obtained by using a resin for peptidesynthesis which is suited for amidation. The resin includes chloromethylresin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin,4-benzyloxybenzyl alcohol resin, 4-methylbenz-hydrylamine resin, PAMresin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamideresin, 4-(2′,4′-dimethoxyphenyl-hydroxymethyl)phenoxy resin,4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin, 2-chlorotritylchloride resin, and so on. Using such a resin, amino acids whose α-aminogroups and functional groups of side-chain have been suitably protectedare condensed on the resin according to the sequence of the objectivepeptide by various condensation techniques which are known per se. Atthe end of the series of reactions, the peptide or the protected peptideis removed from the resin and the protective groups are removed and ifnecessary, disulfide bonds are formed to obtain the objectivepolypeptide.

For the condensation of the above-mentioned protected amino acids, avariety of activating reagents for peptide synthesis can be used such asHATU, HCTU or e.g. a carbodiimide. The carbodiimide includes DCC,N,N′-diisopropylcarbodiimide, andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide. For activation with sucha reagent, a racemization inhibitor additive, e.g. HOBt or Oxyma Purecan be used. The protected amino acid can be directly added to the resinalong with the activation reagents and racemization inhibitor or bepre-activated as symmetric acid anhydride, HOBt ester, or HOOBt esterthen added to the resin. The solvent for the activation of protectedamino acids or condensation with the resin can be properly selected fromamong those solvents which are known to be useful for peptidecondensation reactions. For example, N,N-dimethylformamide,N-methylpyrrolidone, chloroform, trifluoroethanol, dimethyl sulfoxide,DMF, pyridine, dioxane, methylene chloride, tetrahydrofuran,acetonitrile, ethyl acetate, or suitable mixtures of them can bementioned. The reaction temperature can be selected from the rangehitherto-known to be useful for peptide bond formation and is usuallyselected from the range of about −20° C.-50° C. The activated amino acidderivative is generally used in a proportion of 1.5-4 fold excess. Ifthe condensation is found to be insufficient by a test utilizing theninhydrin reaction, the condensation reaction can be repeated to achievea sufficient condensation without removing the protective group. Ifrepeated condensation still fails to provide a sufficient degree ofcondensation, the unreacted amino group can be acetylated with aceticanhydride or acetylimidazole.

The protecting group of amino group for the starting material amino acidincludes Z, Boc, tertiary-amyloxycarbonyl, isobornyloxycarbonyl,4-methoxybenzyloxycarbonyl, Cl—Z, Br—Z, adamantyloxycarbonyl,trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulfenyl,diphenylphosphinothioyl, or Fmoc. The carboxy-protecting group that canbe used includes but is not limited to the above-mentioned C₁₋₆ alkyl,C₃₋₈ cycloalkyl and C₆₋₁₀aryl-C₁₋₂alkyl as well as 2-adamantyl,4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, phenacyl,benzyloxycarbonylhydrazido, tertiary-butoxycarbonylhydrazido, andtritylhydrazido.

The hydroxy group of serine and threonine can be protected byesterification or etherification. The group suited for saidesterification includes carbon-derived groups such as lower alkanoylgroups, e.g. acetyl etc., aroyl groups, e.g. benzoyl etc.,benzyloxycarbonyl, and ethoxycarbonyl. The group suited for saidetherification includes benzyl, tetrahydropyranyl, and tertiary-butyl.The protective group for the phenolic hydroxyl group of tyrosineincludes Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br—Z, and tertiary-butyl.

The protecting group of imidazole for histidine includes Tos,4-methoxy-2,3,6-tri ethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum,Boc, Trt, and Fmoc.

The activated carboxyl group of the starting amino acid includes thecorresponding acid anhydride, azide and active esters, e.g. esters withalcohols such as pentachlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB,N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt, etc. The activatedamino group of the starting amino acid includes the correspondingphosphoramide.

The method for elimination of protective groups includes catalyticreduction using hydrogen gas in the presence of a catalyst such aspalladium black or palladium-on-carbon, acid treatment with anhydroushydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid,trifluoroacetic acid, or a mixture of such acids, base treatment withdiisopropylethylamine, triethylamine, piperidine, piperazine, reductionwith sodium metal in liquid ammonia. The elimination reaction by theabove-mentioned acid treatment is generally carried out at a temperatureof −20° C.-40° C. and can be conducted advantageously with addition of acation acceptor such as anisole, phenol, thioanisole, m-cresol,p-cresol, dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol. The2,4-dinitrophenyl group used for protecting the imidazole group ofhistidine can be eliminated by treatment with thiophenol, while theformyl group used for protecting the indole group of tryptophan can beeliminated by alkali treatment with dilute sodium hydroxide solution ordilute aqueous ammonia as well as the above-mentioned acid treatment inthe presence of 1,2-ethanedithiol, 1,4-butanedithiol.

The method for protecting functional groups which should not take partin the reaction of the starting material, the protective groups that canbe used, the method of removing the protective groups, and the method ofactivating the functional groups that are to take part in the reactioncan all be selected judicially from among the known groups and methods.

An another method for obtaining the amide form of the polypeptidecomprises amidating the -carboxyl group of the C-terminal amino acid atfirst, then extending the peptide chain to the N-side until the desiredchain length, and then selectively deprotecting the α-amino group of theC-terminal peptide and the α-carboxy group of the amino acid or peptidethat is to form the remainder of the objective polypeptide andcondensing the two fragments whose α-amino group and side-chainfunctional groups have been protected with suitable protective groupsmentioned above in a mixed solvent such as that mentioned hereinbefore.The parameters of this condensation reaction can be the same asdescribed hereinbefore. From the protected peptide obtained bycondensation, all the protective groups are removed by theabove-described method to thereby provide the desired crude peptide.This crude peptide can be purified by known purification procedures andthe main fraction be lyophilized to provide the objective amidatedpolypeptide. To obtain an ester of the polypeptide, the α-carboxyl groupof the C-terminal amino acid is condensed with a desired alcohol to givean amino acid ester and then, the procedure described above forproduction of the amide is followed.

Alternatively, recombinant expression methods are particularly useful.Recombinant protein expression using a host cell (a cell artificiallyengineered to comprise nucleic acids encoding the sequence of thepeptide and which will transcribe and translate, and optionally, secretethe peptide into the cell growth medium) is used routinely in the art.For recombinant production process, a nucleic acid coding for amino acidsequence of the peptide would typically be synthesized by conventionallymethods and integrated into an expression vector. Such methods isparticularly preferred for manufacture of the polypeptide compositionscomprising the peptides fused to additional peptide sequences or otherproteins or protein fragments or domains. The host cell can optionallybe at least one selected from from E. Coli, COS-1, COS-7, HEK293, BHT21,CHO, BSC-1, Hep G2, 653, SP2/0, 293, heLa, myeloma, lymphoma, yeast,insect or plant cells, or any derivative, immortalized or transformedcell thereof.

The invention also encompasses polynucleotides encoding theabove-described variants that may be in the form of RNA or in the formof DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNAmay be double-stranded or single-stranded. The coding sequences thatencode the compositions of the present invention may vary as a result ofthe redundancy or degeneracy of the genetic code.

The polynucleotides that encode for the compositions of the presentinvention may include the following: only the coding sequence for thevariant, the coding sequence for the variant and additional codingsequence such as a functional polypeptide, or a leader or secretorysequence or a pro-protein sequence; the coding sequence for the variantand non-coding sequence, such as introns or non-coding sequence 5′and/or 3′ of the coding sequence for the variant. Thus the term“polynucleotide encoding a variant” encompasses a polynucleotide thatmay include not only coding sequence for the variant but also apolynucleotide, which includes additional coding and/or non-codingsequence.

The invention further relates to variants of the describedpolynucleotides that encode for fragments, analogs and derivatives ofthe polypeptide that contain the indicated substitutions. The variant ofthe polynucleotide may be a naturally occurring allelic variant of thehuman GDF15 sequence, a non-naturally occurring variant, or a truncatedvariant as described above. Thus, the present invention also includespolynucleotides encoding the variants described above, as well asvariants of such polynucleotides, which variants encode for a fragment,derivative or analog of the disclosed variant. Such nucleotide variantsinclude deletion variants, substitution variants, truncated variants,and addition or insertion variants as long as at least one of theindicated amino acid substitutions of the first or second embodiments ispresent.

The polynucleotides of the invention can be expressed in hosts after thesequences have been operably linked to (i.e., positioned to ensure thefunctioning of) an expression control sequence. These expression vectorsare typically replicable in the host organisms either as episomes or asan integral part of the host chromosomal DNA. Commonly, expressionvectors will contain selection markers, e.g., tetracycline, neomycin,and dihydrofolate reductase, to permit detection of those cellstransformed with the desired DNA sequences. The GDF15 variant can beexpressed in mammalian cells, insect, yeast, bacterial or other cellsunder the control of appropriate promoters. Cell free translationsystems can also be employed to produce such proteins using RNAs derivedfrom DNA constructs of the present invention.

Escherichia Coli (E. coli) is a prokaryotic host useful particularly forcloning the polynucleotides of the present invention. Other microbialhosts suitable for use include Bacillus subtilus, Salmonellatyphimurium, and various species of Serratia, Pseudomonas,Streptococcus, and Staphylococcus, although others may also be employedas a matter of choice. In these prokaryotic hosts, one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any of a number of well-known promoters maybe present, such as the lactose promoter system, a tryptophan (Trp)promoter system, a beta-lactamase promoter system, or a promoter systemfrom phages lambda or T7. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

One skilled in the art of expression of proteins will recognize thatmethionine or methionine-arginine sequence can be introduced at theN-terminus of the mature sequence for expression in E. coli and arecontemplated within the context of this invention. Thus, unlessotherwise noted, compositions of the present invention expressed in E.coli have a methionine sequence introduced at the N-terminus.

Other microbes, such as yeast or fungi, may also be used for expression.Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe,and Pichia angusta are examples of preferred yeast hosts, with suitablevectors having expression control sequences, such as promoters,including 3-phosphoglycerate kinase or other glycolytic enzymes, and anorigin of replication, termination sequences and the like as desired.Aspergillus niger, Trichoderma reesei; and Schizophyllum commune, areexamples of fungi hosts, although others may also be employed as amatter of choice.

Mammalian tissue cell culture may also be used to express and producethe polypeptides of the present invention. A number of suitable hostcell lines capable of secreting intact variants have been developed inthe art, and include the CHO cell lines, various COS cell lines, NSOcells, Syrian Hamster Ovary cell lines, HeLa cells, or human embryonickidney cell lines (i.e. HEK293, HEK293EBNA).

Expression vectors for mammalian cells can include expression controlsequences, such as an origin of replication, a promoter, an enhancer,and necessary processing information sites, such as ribosome bindingsites, RNA splice sites, polyadenylation sites, and transcriptionalterminator sequences. Preferred expression control sequences arepromoters derived from SV40, adenovirus, bovine papilloma virus,cytomegalovirus, Raus sarcoma virus, and the like. Preferredpolyadenylation sites include sequences derived from SV40 and bovinegrowth hormone.

The vectors containing the polynucleotide sequences of interest (e.g.,that encode the compositions of the present invention and expressioncontrol sequences) can be transferred into the host cell by well-knownmethods, which vary depending on the type of cellular host. For example,calcium chloride transfection is commonly utilized for prokaryoticcells, whereas calcium phosphate treatment or electroporation may beused for other cellular hosts.

Various methods of protein purification may be employed and such methodsare known in the art and described, for example, in Deutscher, Methodsin Enzymology 182: 83-9 (1990) and Scopes, Protein Purification:Principles and Practice, Springer-Verlag, NY (1982). The purificationstep(s) selected will depend, for example, on the nature of theproduction process used for the compositions of the present invention.

The polypeptides may be prepared in substantially pure or isolated form(e.g., free from other polypeptides). The polypeptides can be present ina composition that is enriched for the polypeptide relative to othercomponents that may be present (e.g., other polypeptides or other hostcell components). For example, purified polypeptide may be provided suchthat the polypeptide is present in a composition that is substantiallyfree of other expressed proteins, e.g., less than 90%, less than 60%,less than 50%, less than 40%, less than 30%, less than 20%, less than10%, less than 5%, or less than 1%, of the composition is made up ofother expressed proteins

Synthesis of Fatty Acid Moiety

Scheme 1 describes the synthesis of a fatty acid moiety of Formula A2.

wherein P¹ and P² are carboxylic acid protective group such as forexample methyl, ethyl, tert-butyl, methoxybenzyl, benzyl, benzyloxy,methoxymethyl, methylthiomethyl, tetrahydropyranyl, phenacyl,N-Phthalimide, cinnamyl, triphenylmethyl, 9-anthrylmethyl, piperonyl,trimethylsilyl, t-butyldimethylsilyl or 2-alkyl 1,3 oxazolines; whereinLG is a leaving group such as for example halo (e.g. Br, Cl, I) ortrifluoromethanesulfonyloxy and wherein R⁴ and p are as described inembodiment 1.

Alkylation of protected malonic acid (1A) with an alkylating agent (1B)in the presence of a base (e.g. sodium hydride, potassium or cesiumcarbonates, sodium hydroxide, lithium diisopropyl amide, sodiumbis(trimethylsilyl)amide, potassium bis(trimethylsilyl)amide, lithiumtertamethylpiperidide, 1,8-Diaazacycloundec-7-ene, N,N-diisopropyl ethylamine or 2,6-dit-butylputridine), in a solvent such as DMF, THF ordimethyl acetamide, generates the protected fatty acid moiety (1C). WhenR⁴ is OH or CO₂H, protection of these functional groups may be requiredprior to the alkylation step. Protective groups for hydroxyl are knownin the art and are for example 1. ethers such as Methyl ether,methoxymethyl ether (MOM), Tetrahydropyranyl ether (THP), t-Butyl ether,allyl ether, benzyl ether, t-butyldimathylsilyl ether, t-butyldiphenylsilyl ether, tribenzyl silyl ether, isopropyldimethylsilyl ether,triphenylmethyl ether, nitrobenzyl ether, 2. Esters and carbonates suchas acetic acid ester, formate ester, trichloroacetate ester,phenoxyacetate ester, pivaloate ester, benzoate ester, methyl carbonate,benzyl carbonate, allyl carbonate, nitrate ester, adamanoate ester,notrophenyl carbonate.

The fatty acid moiety of Formula A2 is obtained by deprotection usingappropriate deprotection method. Standard methods can be applied for thehydrolysis of the intermediate (1C) using a base selected from, but notlimited to, NaOH, KOH, or LiOH, or an acid selected from, but notlimited to, TFA, HCl, or BCl₃. When P¹ or P² is benzyl or methoxybenzyl,a preferable method of the deprotection is hydrogenation in the presenceof a catalyst such as, but not limited to, palladium-on-carbon.

Scheme 2 illustrates the synthesis of an fatty acid moiety of Formula A¹wherein R¹ is C(O)₂H.

wherein P¹ and P², LG are as defined supra and R², R³, n and m are asdefined in embodiment 1.

Protected malonic acid (1A) undergoes 2 subsequent alkylations withalkylating agent (2A) and (2C), order of which can be reversed, prior todeprotection using appropriate method as described supra in Scheme 1.When R² and R³ are OH or CO₂H, protection of these functional groups maybe required prior to the alkylation steps.

The fatty acid moiety of Formula A¹ wherein R¹ is H can be prepared bydecarboxylation of the corresponding fatty acid moiety of Formula A¹wherein R¹ is CO2H. Decarboxylation conditions are well known in the artsuch as for example decarboxylation under basic condition (e.g. Ammoniumhydroxide).

Synthesis of Biomolecule-Linker Construct

wherein B is biomolecule or a modified form thereof, Z¹ is is a C₁-C₂₀alkylene linker wherein the alkylene chain is optionally substitutedwith oxo (═O), and wherein one or more carbon is replaced with O or NH;and wherein C1 is a mono, di or tricyclic carbocyclic or heterocyclicring system optionally substituted with fluorine.

The cycloalkyne (3B) is attached to an amino residue of the biomolecule(3A) (for example to the amino functionality of the N-terminus or theside chain of a lysine) via its carboxylic acid reactive group usingstandard amide coupling methods. Known coupling methods may be appliedincluding, but not limited to, conversion of the intermediate (3B) to anactivated form thereof, [e.g. to a corresponding pyrrolidine-2,5-dione(using standard N-hydrosuccinimide chemistry), or converting acid (3B)using reagents such as triphosgene, carbonyldiimidazole, 4-nitrophenylchloroformate, or disuccinimidyl carbonate, conversion of the acid (3B)to a corresponding acid halide, using reagents such as thionyl chlorideor oxalyl chloride, or conversion of the acid (3B) to a correspondingmixed anhydride using reagents such as ClC(O)O-isobutyl,2,4,6-trichlorobenzoyl chloride or propyl phosphonic acid anhydridecyclic trimer (T3P), followed by reaction of the oxazolidine-2,5-dione,the acid halide, or the mixed anhydride] with the biomolecule (3A) in apresence or absence of a base such as tertiary amine (e.g. triethylamineor N,N-diisoproplyl ethylamine) or K₂CO₃. Alternatively, the biomolecule3A can be coupled with the acid 3B using peptide condensation reagentsincluding, but not limited to, dicyclohexylcarbodiimide (DCC),diisopropylcarbodiimide (DIC),1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC HCl),benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP), or benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP) in presence of or absence of a reagent such as1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, ordimethylaminopyridine. Preferably, the cycloalkyne/acid intermediate(3B) is converted to its activated form thereof using NHS chemistryprior to reacting with the amino functionality on the biomolecule.

A selective acylation of the amino functionality at the N-terminus ofthe biomolecule has been developed and reported in a co-filed USapplication Nos. 62/015,858 and 62/082,337.

The selective acylation involves the reaction of NHS activatedcyclooctyne analog (NHS derivatives of (3B) with a biomolecule where theN-terminus has been modified to include a histidine amino acid adjacentto the N-terminus amino acid. The reaction is highly selective for theamino functionality at the N-terminus when carried out at pH 4, due tothe presence of a neighboring effect of the histidine amino acid.

Synthesis of Fatty Acid Residue Linker Construct

Fatty Acid-Linker Construct for Click Chemistry

Scheme 4 describes the synthesis of a fatty acid-PEG linker constructwith a terminal azido functional group.

wherein y is 0 to 34 and FA is an fatty acid moiety as described inFormula A1, A2 or A3 which is attached via one of its carboxylic acidfunctionality to the PEG linker, FA has the following Formulae:

The fatty acid moiety (4B) is attached to a PEG containing linker (4A)via an amide coupling reaction. Known coupling methods have beendescribed in detail supra in Scheme 3. Preferably the acid functionalityon the fatty acid moiety is activated using NHS chemistry.

Where R¹ is CO₂H, R₂, R₃ and R₄ are CO₂H or OH, protecting groups mayneed to be introduced prior to the coupling reaction in order to controlthe reactive site. Protecting group for carboxylic acid and hydroxygroups have been described supra in scheme 1. Alternatively, selectiveactivation of carboxylic acid can be achieved using NHS chemistry.

Fatty Acid-Linker for Direct Attachment to the Biomolecule of Interest

Scheme 5 describes the synthesis of an fatty acid-PEG linker constructwith a terminal CO2H functional group.

wherein FA is as defined supra in Scheme 4 and y is 0 to 34.

The fatty acid (4B) may be attached to a PEG containing linker (5A)using amide coupling described supra.

Fatty Acid-Linker Construct for Attachment to a Biomolecule of InterestUsing Transglutaminase Enzyme

Scheme 5A describes the preparation of a Fatty acid-linker constructcontaining a glutamic acid amino acid allowing for site selectivemodification of a lysine when using transglutaminase enzyme.

wherein y and FA are as previously defined. Such constructs allow forselective site modification of an amino group on the side chain of alysine. This transglutaminase selective site modification of protein hasbeen described in U.S. application No. 61/845,273 filed on Jul. 11,2013.Synthesis of Conjugate of the InventionConjugation Using Click Chemistry

wherein B is a biomolecule of interest or a modified form thereof (forexample mutant or a biomolecule containing a histidine tag) and y, C1,Z₁, FA and y are defined supra.

Cycloalkyne construct (3C) undergoes a Huisgen cycloaddition with aterminal azide of the Fatty acid-linker construct (4C) as commonly knownas click chemistry. Example of click chemistries have been described inUS 2009/0068738.

Conjugation Via Direct Attachment Using Coupling Conditions

wherein B is a biomolecule of interest or a modified form thereof (suchas for example mutant and/or a biomolecule containing a histidine tag)and the fatty acid-linker construct is attached to the N-terminus of thebiomolecule.

The fatty acid-linker construct (5B) is attached to an amino residue ofthe biomolecule (3A) (for example to the amino functionality of theN-terminus or the side chain of a lysine) via its carboxylic acidreactive group using standard amide coupling methods. Known couplingmethods have been described in detail supra in Scheme 3. Preferably theacid functionality on the fatty acid-linker construct is activated usingNHS chemistry.

A selective acylation of the amino functionality at the N-terminus ofthe biomolecule has been developed and reported in a co-filed USapplication Nos. 62/015,858 and 62/082,337. The selective acylationinvolves the reaction of a NHS activated compound (NHS derivatives of(5B)) with a biomolecule where the N-terminus has been modified toinclude a histidine amino acid adjacent to the N-terminus amino acid.The reaction is highly selective for the amino functionality at theN-terminus when carried out at pH 4, due to the presence of aneighboring effect of the histidine amino acid.

Conjugation Using Transglutaminase Enzyme

Selective modification of the biomolecule at its lysine side chain canbe achieved using transglutaminase enzyme. Such modification has beenreported in U.S. application No. 61/845,273 filed Jul. 11, 2013 or WO2015/006728 (in example 25 of this application).Pharmaceutical Composition

The conjugate of the instant invention may be administered in any of avariety of ways, including subcutaneously, intramuscularly,intravenously, intraperitoneally, inhalationally, intranasally, orallyetc. Particularly preferred embodiments of the invention employcontinuous intravenous administration of the conjugates of the instantinvention, or an amide, ester, or salt thereof. The conjugates on theinstant invention may be administered as a bolus or as a continuousinfusion over a period of time. An implantable pump may be used. Incertain embodiments of the invention, intermittent or continuousconjugates administration is continued for one to several days (e.g.,2-3 or more days), or for longer periods of time, e.g., weeks, months,or years. In some embodiments, intermittent or continuous conjugatesadministration is provided for at least about 3 days, preferably atleast about 6 days. In other embodiments, intermittent or continuousconjugate administration is provided for at least about one week. Inother embodiments, intermittent or continuous conjugate administrationis provided for at least about two weeks. It may be desirable tomaintain an average plasma conjugate concentration above a particularthreshold value either during administration or between administrationof multiple doses. A desirable concentration may be determined, forexample, based on the subject's physiological condition, diseaseseverity, etc. Such desirable value(s) can be identified by performingstandard clinical trials. Alternatively, the peptides and conjugatesthereof could be delivered orally via FcRn mechanism. (Nat Rev Immunol.7(9), 715-25, 2007; Nat Commun. 3; 3:610, 2012, Am J PhysiolGastrointest Liver Physiol 304: G262-G270, 2013).

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a conjugate of the present invention or an amide,an ester or a salt thereof and one or more pharmaceutically acceptablecarriers. The pharmaceutical composition can be formulated forparticular routes of administration such as oral administration,subcutaneous administration, parenteral administration, and rectaladministration, etc. In addition, the pharmaceutical compositions of thepresent invention can be made up in a solid form (including withoutlimitation capsules, tablets, pills, granules, lyophilizates, powders orsuppositories), or in a liquid form (including without limitationsolutions, suspensions or emulsions). The pharmaceutical compositionscan be subjected to conventional pharmaceutical operations such asaseptic manufacturing, sterilization and/or can contain conventionalinert diluents, cake forming agents, tonicity agents, lubricatingagents, or buffering agents, as well as adjuvants, such aspreservatives, stabilizers, wetting agents, emulsifers and buffers, etc.

Pharmaceutical compositions suitable for injectable use typicallyinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion.

For intravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the composition should besterile and should be fluid to the extent that easy syringabilityexists. Preferred pharmaceutical formulations are stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Ingeneral, the relevant carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, amino acids, sorbitol, sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent which delays absorption, for example, aluminum monostearate andgelatin. In some embodiments, a multifunctional excipient such asrecombinant albumin may be incorporated into the formulation process tofacilitate the stabilization of the conjugate product from degradationor aggregation, to improve solubility and assist in the administrationand release of the active component. (BioPharm International, 2012, Vol23, Issue 3, pp 40-44).

Certain injectable compositions are aqueous isotonic solutions orsuspensions, and suppositories are advantageously prepared from fattyemulsions or suspensions. Said compositions may be sterilized and/orcontain adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure and/or buffers. In addition, they may also contain othertherapeutically valuable substances. Said compositions are preparedaccording to conventional mixing, granulating or coating methods,respectively, and contain about 0.1-75%, or contain about 1-50%, of theactive ingredient.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltration sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. Formulations fororal delivery may advantageously incorporate agents to improve stabilitywithin the gastrointestinal tract and/or to enhance absorption.

For administration by inhalation, the inventive therapeutic agents arepreferably delivered in the form of an aerosol spray from pressuredcontainer or dispenser which contains a suitable propellant, e.g., a gassuch as carbon dioxide, or a nebulizer. It is noted that the lungsprovide a large surface area for systemic delivery of therapeuticagents.

The agents may be encapsulated, e.g., in polymeric microparticles suchas those described in U.S. publication 20040096403, or in associationwith any of a wide variety of other drug delivery vehicles that areknown in the art. In other embodiments of the invention the agents aredelivered in association with a charged lipid as described, for example,in U.S. publication 20040062718. It is noted that the latter system hasbeen used for administration of a therapeutic polypeptide, insulin,demonstrating the utility of this system for administration of peptideagents.

Systemic administration can also be by transmucosal or transdermalmeans.

Suitable compositions for transdermal application include an effectiveamount of a conjugate of the invention with a suitable carrier. Carrierssuitable for transdermal delivery include absorbable pharmacologicallyacceptable solvents to assist passage through the skin of the host. Forexample, transdermal devices are in the form of a bandage comprising abacking member, a reservoir containing the compound optionally withcarriers, optionally a rate controlling barrier to deliver the compoundof the skin of the host at a controlled and predetermined rate over aprolonged period of time, and means to secure the device to the skin.

Suitable compositions for topical application, e.g., to the skin andeyes, include aqueous solutions, suspensions, ointments, creams, gels orsprayable formulations, e.g., for delivery by aerosol or the like. Suchtopical delivery systems will in particular be appropriate for dermalapplication. They are thus particularly suited for use in topical,including cosmetic, formulations well-known in the art. Such may containsolubilizers, stabilizers, tonicity enhancing agents, buffers andpreservatives.

In certain embodiments, the pharmaceutical composition is forsubcutaneous administration. Suitable formulation components and methodsfor subcutaneous administration of polypeptide therapeutics (e.g.,antibodies, fusion proteins and the like) are known in the art. See,e.g., Published United States Patent Application No 2011/0044977 andU.S. Pat. Nos. 8,465,739 and 8,476,239. Typically, the pharmaceuticalcompositions for subcutaneous administration contain suitablestabilizers (e.g, amino acids, such as methionine, and or saccharidessuch as sucrose), buffering agents and tonicifying agents.

As used herein a topical application may also pertain to an inhalationor to an intranasal application. They may be conveniently delivered inthe form of a dry powder (either alone, as a mixture, for example a dryblend with lactose, or a mixed component particle, for example withphospholipids) from a dry powder inhaler or an aerosol spraypresentation from a pressurised container, pump, spray, atomizer ornebuliser, with or without the use of a suitable propellant.

The invention further provides pharmaceutical compositions and dosageforms that comprise one or more agents that reduce the rate by which thecompound of the present invention as an active ingredient willdecompose. Such agents, which are referred to herein as “stabilizers,”include, but are not limited to, antioxidants such as ascorbic acid, pHbuffers, or salt buffers, recombinant Albumin.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts that retain the biological effectiveness and properties of theconjugates of this invention and, which typically are not biologicallyor otherwise undesirable. In many cases, the conjugates of the presentinvention are capable of forming acid and/or base salts by virtue of thepresence of amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids, e.g., acetate, aspartate, benzoate,besylate, bromide/hydrobromide, bicarbonate/carbonate,bisulfate/sulfate, camphorsulfornate, chloride/hydrochloride,chlortheophyllonate, citrate, ethandisulfonate, fumarate, gluceptate,gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate,lactate, lactobionate, laurylsulfate, malate, maleate, malonate,mandelate, mesylate, methylsulphate, naphthoate, napsylate, nicotinate,nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, polygalacturonate,propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate andtrifluoroacetate salts.

Inorganic acids from which salts can be derived include, for example,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Organic acids from which salts can bederived include, for example, acetic acid, propionic acid, glycolicacid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid,sulfosalicylic acid, and the like. Pharmaceutically acceptable baseaddition salts can be formed with inorganic and organic bases.

Inorganic bases from which salts can be derived include, for example,ammonium salts and metals from columns I to XII of the periodic table.In certain embodiments, the salts are derived from sodium, potassium,ammonium, calcium, magnesium, iron, silver, zinc, and copper;particularly suitable salts include ammonium, potassium, sodium, calciumand magnesium salts.

Organic bases from which salts can be derived include, for example,primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, basic ionexchange resins, and the like. Certain organic amines includeisopropylamine, benzathine, cholinate, diethanolamine, diethylamine,lysine, meglumine, piperazine and tromethamine.

The pharmaceutically acceptable salts of the present invention can besynthesized from a parent compound, a basic or acidic moiety, byconventional chemical methods. Generally, such salts can be prepared byreacting free acid forms of these compounds with a stoichiometric amountof the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate,bicarbonate or the like), or by reacting free base forms of thesecompounds with a stoichiometric amount of the appropriate acid. Suchreactions are typically carried out in water or in an organic solvent,or in a mixture of the two. Generally, use of non-aqueous media likeether, ethyl acetate, ethanol, isopropanol, or acetonitrile isdesirable, where practicable. Lists of additional suitable salts can befound, e.g., in “Remington's Pharmaceutical Sciences”, 20th ed., MackPublishing Company, Easton, Pa., (1985); and in “Handbook ofPharmaceutical Salts: Properties, Selection, and Use” by Stahl andWermuth (Wiley-VCH, Weinheim, Germany, 2002).

As used herein, the term “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, multifunctional excipient such as recombinant albumin and the likeand combinations thereof, as would be known to those skilled in the art(see, for example, Remington's Pharmaceutical Sciences, 18th Ed. MackPrinting Company, 1990, pp. 1289-1329). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the therapeutic or pharmaceutical compositions is contemplated.

Method of the Invention

GDF15 circulating levels have been reported to be elevated in multiplepathological and physiological conditions, most notably pregnancy (MooreAG 2000. J Clin Endocrinol Metab 85: 4781-4788), β-thalassemia (Tanno T2007, Nat Med 13:1096-101) (Zimmermann M B, 2008 Am J Clin Nutr88:1026-31), and congenital dyserythropoietic anemia (Tamary H 2008,Blood. 112:5241-4). GDF15 has also been linked to multiple biologicalactivities in literature reports. Studies of GDF15 knockout andtransgenic mice suggested that GDF15 may be protective againstischemic/reperfusion- or overload-induced heart injury (Kempf T, 2006,Circ Res. 98:351-60) (Xu J, 2006, Circ Res. 98:342-50), protectiveagainst aging-associated motor neuron and sensory neuron loss (StrelauJ, 2009, J Neurosci. 29: 13640-8), mildly protective against metabolicacidosis in kidney, and may cause cachexia in cancer patients (Johnen H2007 Nat Med. 11: 1333-40). Many groups also studied the role of GDF15in cell apoptosis and proliferation and reported controversial resultsusing different cell culture and xenograft models. Studies on transgenicmice showed that GDF15 is protective against carcinogen or Ape mutationinduced neoplasia in intestine and lung (Baek S J 2006,Gastroenterology. 131: 1553-60; Cekanova M 2009, Cancer Prev Res2:450-8).

GDF15 has also been reported to play a role in inflammation, cancer andmetabolism (Samule Breit et al. Growth Factors, October 2011; 29(5):187-195). GDF15 has further been implicated in the regulation ofphysiological appetite and body weight (Vicky Wang-Wei Tsai et al.Public Library of Science: PLOS ONE 2013, Vol. 8, Issue 2, e55174)

The present invention provides methods for treating or preventingmetabolic disorders or diseases, diabetes, type 2 diabetes mellitus,obesity, pancreatitis, dyslipidemia, alcoholic and nonalcoholic fattyliver disease/steatohepatitis and other progressive liver diseases,insulin resistance, hyperinsulinemia, glucose intolerance,hyperglycemia, metabolic syndrome, hypertension, cardiovascular disease,atherosclerosis, peripheral arterial disease, stroke, heart failure,coronary heart disease, diabetic complications (including but notlimited to chronic kidney disease), neuropathy, gastroparesis and othermetabolic disorders, in a subject in need thereof, comprising:administering to the subject a therapeutically effective amount of aconjugate of the invention, or an amide, ester or salt thereof or amixture of conjugates, wherein the biomolecule is human GrowthDifferentiation Factor 15 (GDF15), homologs, variants, mutants,fragments and other modified forms thereof.

Such methods may have an advantageous effect such as for exampledecreasing the frequency of administration.

Thus, as a further embodiment, the present invention provides the use ofa conjugate as described herein, or an amide, ester or apharmaceutically acceptable salt thereof or a mixture of the conjugatesdescribed therein, wherein the biomolecule is human GrowthDifferentiation Factor 15 (GDF15), homologs, variants, mutants,fragments and other modified forms thereof, for the treatment ofmetabolic disorders or diseases, type 2 diabetes mellitus, obesity,pancreatitis, dyslipidemia, alcoholic and nonalcoholic fatty liverdisease/steatohepatitis and other progressive liver diseases, insulinresistance, hyperinsulinemia, glucose intolerance, hyperglycemia,metabolic syndrome, hypertension, cardiovascular disease,atherosclerosis, peripheral arterial disease, stroke, heart failure,coronary heart disease, diabetic complications (including but notlimited to chronic kidney disease), neuropathy, gastroparesis and othermetabolic disorders.

Thus, as a further embodiment, the present invention provides the use ofa conjugate or an amide, an ester or a pharmaceutically acceptable saltthereof, or a mixture of conjugates, in therapy.

The effective amount of a pharmaceutical composition or combination ofthe invention to be employed therapeutically will depend, for example,upon the therapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment will thusvary depending, in part, upon the molecule delivered, the indication forwhich the conjugate is being used, the route of administration, and thesize (body weight, body surface, or organ size) and condition (the ageand general health) of the patient. Accordingly, the clinician can titerthe dosage and modify the route of administration to obtain the optimaltherapeutic effect. A typical dosage can range from about 0.1 μg/kg toup to about 100 mg/kg or more, depending on the factors mentioned above.In other embodiments, the dosage can range from 0.1 μg/kg up to about100 mg/kg; or 1 μg/kg up to about 100 mg/kg. In a further aspect of thisembodiment, the dosage can range from 5 μg/kg to 25 μg/kg. In yet afurther aspect of this embodiment, the dosage can range from 10 μg/kg to20 μg/kg.

The frequency of dosing will depend upon the pharmacokinetic parametersof the dual function protein in the formulation being used. Typically, aclinician will administer the composition until a dosage is reached thatachieves the desired effect. The composition can therefore beadministered as a single dose, as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages can be ascertained through use ofappropriate dose-response data.

The terms “therapeutically effective dose” and “therapeuticallyeffective amount,” as used herein, means an amount of conjugate thatelicits a biological or medicinal response in a tissue system, animal,or human being sought by a researcher, physician, or other clinician,which includes alleviation or amelioration of the symptoms of thedisease or disorder being treated, i.e., an amount of GDF15 (or GDF15mutant) polypeptide conjugate that supports an observable level of oneor more desired biological or medicinal response, for example loweringblood glucose, insulin, triglyceride, or cholesterol levels; reducingbody weight; reducing food intake or improving glucose tolerance, energyexpenditure, or insulin sensitivity).

The terms “patient” or “subject” are used interchangeably to refer to ahuman or a non-human animal (e.g., a mammal).

As used herein, the term “treat”, “treating” or “treatment” of anydisease or disorder refers in one embodiment, to ameliorating thedisease or disorder (i.e., slowing or arresting or reducing thedevelopment of the disease or at least one of the clinical symptomsthereof). In another embodiment “treat”, “treating” or “treatment”refers to alleviating or ameliorating at least one physical parameterincluding those which may not be discernible by the patient. In yetanother embodiment, “treat”, “treating” or “treatment” refers tomodulating the disease or disorder, either physically, (e.g.,stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both. Thus, treatmentincludes inhibiting (i.e., arresting the development or furtherdevelopment of the disease, disorder or condition or clinical symptomsassociation therewith) an active disease (e.g. for example in the caseof GDF15 conjugate, so as to decrease body weight, to decrease foodintake, to decrease the level of insulin and/or glucose in thebloodstream, to increase glucose tolerance so as to minimize fluctuationof glucose levels, and/or so as to protect against diseases caused bydisruption of glucose homeostasis).

In yet another embodiment, “treat”, “treating” or “treatment” refers topreventing or delaying the onset or development or progression of thedisease or disorder.

The term “in need of treatment” as used herein refers to a judgment madeby a physician or other caregiver that a subject requires or willbenefit from treatment. This judgment is made based on a variety offactors that are in the realm of the physician's or caregiver'sexpertise.

The terms “prevent”, “preventing”, “prevention” and the like refer to acourse of action (such as administering a conjugate of the invention ora pharmaceutical composition comprising a conjugate) initiated in amanner (e.g., prior to the onset of a disease, disorder, condition orsymptom thereof) so as to prevent, suppress, inhibit or reduce, eithertemporarily or permanently, a subject's risk of developing a disease,disorder, condition or the like (as determined by, for example, theabsence of clinical symptoms) or delaying the onset thereof, generallyin the context of a subject predisposed to having a particular disease,disorder or condition. In certain instances, the terms also refer toslowing the progression of the disease, disorder or condition orinhibiting progression thereof to a harmful or otherwise undesiredstate.

The term “metabolic disease or disorder” refers to an associated clusterof traits that includes, but is not limited to, hyperinsulinemia,abnormal glucose tolerance, obesity, redistribution of fat to theabdominal or upper body compartment, hypertension, dyslipidemiacharacterized by high triglycerides, low high density lipoprotein(HDL)-cholesterol, and high small dense low density lipoprotein (LDL)particles. Subjects having metabolic disease or disorder are at risk fordevelopment of Type 2 diabetes and, for example, atherosclerosis.

The phrase “glucose metabolism disorder” encompasses any disordercharacterized by a clinical symptom or a combination of clinicalsymptoms that is associated with an elevated level of glucose and/or anelevated level of insulin in a subject relative to a healthy individual.Elevated levels of glucose and/or insulin may be manifested in thefollowing diseases, disorders and conditions: hyperglycemia, type IIdiabetes, gestational diabetes, type I diabetes, insulin resistance,impaired glucose tolerance, hyperinsulinemia, impaired glucosemetabolism, pre-diabetes, metabolic disorders (such as metabolic diseaseor disorder, which is also referred to as syndrome X), and obesity,among others. The GDF15 conjugates of the present disclosure, andcompositions thereof, can be used, for example, to achieve and/ormaintain glucose homeostasis, e.g., to reduce glucose level in thebloodstream and/or to reduce insulin level to a range found in a healthysubject.

The term “insulin resistance” as used herein refers to a condition wherea normal amount of insulin is unable to produce a normal physiologicalor molecular response. In some cases, a hyper-physiological amount ofinsulin, either endogenously produced or exogenously administered, isable to overcome the insulin resistance, in whole or in part, andproduce a biologic response.

The phrase “glucose tolerance”, as used herein, refers to the ability ofa subject to control the level of plasma glucose and/or plasma insulinwhen glucose intake fluctuates. For example, glucose toleranceencompasses the subject's ability to reduce, within about 120 minutes,the level of plasma glucose back to a level determined before the intakeof glucose.

The term “Glucose intolerance, or ‘Impaired Glucose Tolerance (IGT) is apre-diabetic state of dysglycemia that is associated with increased riskof cardiovascular pathology. The pre-diabetic condition prevents asubject from moving glucose into cells efficiently and utilizing it asan efficient fuel source, leading to elevated glucose levels in bloodand some degree of insulin resistance.

The term “Type 2 diabetes Mellitus” is a condition characterized byexcess glucose production and circulating glucose levels remainexcessively high as a result of inadequate glucose clearance and theinability of the pancreas to produce enough insulin.

The term “hyperglycemia”, as used herein, refers to a condition in whichan elevated amount of glucose circulates in the blood plasma of asubject relative to a healthy individual. Hyperglycemia can be diagnosedusing methods known in the art, including measurement of fasting bloodglucose levels as described herein.

The term “Hypoglycemia”, also called low blood sugar, occurs when bloodglucose level drops too low to provide enough energy for the body'sactivities.

The term “hyperinsulinemia”, as used herein, refers to a condition inwhich there are elevated levels of circulating insulin when,concomitantly, blood glucose levels are either elevated or normal.Hyperinsulinemia can be caused by insulin resistance which is associatedwith dyslipidemia such as high triglycerides, high cholesterol, highlow-density lipoprotein (LDL) and low high-density lipoprotein (HDL);high uric acids levels; polycystic ovary syndrome; type II diabetes andobesity. Hyperinsulinemia can be diagnosed as having a plasma insulinlevel higher than about 2 pU/mL.

The term “Pancreatitis” is inflammation of the pancreas.

The term “Dyslipidemia” is a disorder of lipoprotein metabolism,including lipoprotein overproduction or deficiency. Dyslipidemias may bemanifested by elevation of the total cholesterol, low-densitylipoprotein (LDL) cholesterol and triglyceride concentrations, and adecrease in high-density lipoprotein (HDL) cholesterol concentration inthe blood.

The term “Fatty liver disease (FLD)”, also known as fatty liver, is acondition wherein large vacuoles of triglyceride fat accumulate in livercells via the process of steatosis (i.e., abnormal retention of lipidswithin a cell). Despite having multiple causes, fatty liver can beconsidered a single disease that occurs worldwide in those withexcessive alcohol intake and the obese (with or without effects ofinsulin resistance insulin). When this process of fat metabolism isdisrupted, the fat can accumulate in the liver in excessive amounts,thus resulting in a fatty liver. Accumulation of fat may also beaccompanied by a progressive inflammation of the liver (hepatitis),called steatohepatitis. By considering the contribution by alcohol,fatty liver may be termed alcoholic steatosis or nonalcoholic fattyliver disease nonalcoholic fatty liver disease (NAFLD), and the moresevere forms as alcoholic steatohepatitis and non-alcoholicsteatohepatitis (NASH).

The term “steatohepatitis” is a type of liver disease, characterized byfatty change of hepatocytes, accompanied by intralobular inflammationand fibrosis. When not associated with excessive alcohol intake, it isreferred to as Nonalcoholic steatohepatitis (NASH).

The term “progressive liver disease” is a liver disease caused by a widerange of liver pathologies that progress from a relatively benign statelike hepatic steatosis to more severe states including hepatitis,fibrosis, cirrhosis, and hepatocellular carcinoma. PNPLA3 has beenspecifically associated with the progressive liver diseases such asNAFLD/NASH, AFLD/ASH, viral hepatitis, Wilson's disease, hereditaryhemochromatosis and primary sclerosing cholangitis (Paola Dongiovanni etal. World Journal of Gastroenterology, 2013, 19(41), 6969-6978)

The term “Obesity,” in terms of the human subject, can be defined as anadult with a Body Mass Index (BMI) of 30 or greater (Centers for DiseaseControl and Prevention). “Metabolic syndrome” can be defined as acluster of risk factors that raises the risk for heart disease and otherdiseases like diabetes and stroke. These risk factors include: highblood sugar—at least 110 milligrams per deciliter (mg/dl) after fasting;high triglycerides—at least 150 mg/dL in the bloodstream; low HDL—lessthan 40 mg/dl; and, blood pressure of 130/85 mmHg or higher (WorldHealth Organization).

The term “Cardiovascular diseases” are diseases related to the heart orblood vessels.

The term “Atherosclerosis” is a vascular disease characterized byirregularly distributed lipid deposits in the intima of large andmedium-sized arteries, sometimes causing narrowing of arterial lumensand proceeding eventually to fibrosis and calcification. Lesions areusually focal and progress slowly and intermittently. Limitation ofblood flow accounts for most clinical manifestations, which vary withthe distribution and severity of lesions.

The term “Coronary heart disease”, also called coronary artery disease,is a narrowing of the small blood vessels that supply blood and oxygento the heart.

“Diabetic complications” are problems caused by high blood glucoselevels, with other body functions such as kidneys, nerves(neuropathies), feet (foot ulcers and poor circulation) and eyes (e.g.retinopathies). Diabetes also increases the risk for heart disease andbone and joint disorders. Other long-term complications of diabetesinclude skin problems, digestive problems, sexual dysfunction andproblems with teeth and gums.

As used herein, the phrase “body weight disorder” refers to conditionsassociated with excessive body weight and/or enhanced appetite. Variousparameters are used to determine whether a subject is overweightcompared to a reference healthy individual, including the subject's age,height, sex and health status. For example, a subject may be consideredoverweight or obese by assessment of the subject's Body Mass Index(BMI), which is calculated by dividing a subject's weight in kilogramsby the subject's height in meters squared. An adult having a BMI in therange of −18.5 to −24.9 kg/m is considered to have a normal weight; anadult having a BMI between −25 and −29.9 kg/m may be consideredoverweight (pre-obese); an adult having a BMI of −30 kg/m or higher maybe considered obese. Enhanced appetite frequently contributes toexcessive body weight. There are several conditions associated withenhanced appetite, including, for example, night eating syndrome, whichis characterized by morning anorexia and evening polyphagia oftenassociated with insomnia, but which may be related to injury to thehypothalamus.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided herein is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionotherwise claimed.

The activity and plasma stability of a conjugate according to thepresent invention can be assessed by the following methods describedbelow.

Assays and Data

The activity and plasma stability of the GDF15 conjugates of Examples 1and 19B according to the present invention can be assessed by thefollowing in vitro and in vivo methods described below.

Methods for Animal Studies

All animal studies described in this document were approved by theNovartis Institutes for Biomedical Research Animal Care and UseCommittee in accordance with local and federal regulations andguidelines. Diet-induced obese male mice (C57BL/6NTac) were purchasedfrom Taconic and fed a 60% fat diet (Research Diets D12492i) from6-weeks of age onward. Upon arrival, mice were housed one animal percage under a 12 h:12 h reverse light-dark cycle. Animals all received aminimum of 1 week acclimation prior to any use. Mice were typicallystudied between 3-4 months of age. One day prior to being studied, micewere randomized based on body weight such that each group had a similaraverage body weight. On the day of study, mice were placed in freshcages, and the old food removed. Approximately 1 h later and just priorto the dark cycle, mice received a single subcutaneous dose of eithervehicle (30 mM sodium acetate, pH 4) or a lipid conjugated GDF15 analog(0.5 mg/kg). After all injections are completed, the mice were reweighedand a defined amount of food returned (˜50 g per mouse). Food intake andbody weight were measured over the course of ˜2 weeks at the timesindicated in the figures. In surrogate animals treated as describedabove, plasma was collected at the indicated times, and GDF15 levelswere measured by ELISA as per the manufacturer's instructions (R&DSystems Quantikine Human GDF15 Immunoassay; DGD150).DIO Mice Single 0.5 mg/kg Sc Dose

The activity and half-life of the conjugates of the invention weretested in the assay described supra.

TABLE 1 Duration of action Body weight Food Intake (BW) Conjugate PK (½life) (FI) reduction reduction (example) (hrs) (days) (days)  1 36 6 8 2 8 8  4 15.1 3 3  5 33.1 8 8  6 3 3  7 21.8 6 6 12 6-8  6-8 13 45.88-10 10 15* 2 6 16 6 6 18 55 8-10 10 19A 8 10 19B crude 86.4 8 14 19B156.9 (exp 1) 14 (exp 1) 14 (exp 1) 50.9 (exp 2) 14 (exp 2) 14 (exp 2)19B2 97.68 (exp 1)   8 (exp 1) 10 (exp 1) 74.2 (exp 2) 14 (exp 2) 14(exp 2) 19B3 98.9 8 10 19Bm 65.04 14  17 Ref ex. 2 3 3 Ref ex. 1 1 1hGDF15 1 1 1 *Lean mice Exp 1: in vivo experiment 1; Exp 2: in vivoexperiment 2.

The data in table 1 demonstrate that the conjugates of the inventionpossess a significant longer duration of action as compared tonon-conjugated hGDF15 and/or as compared to pegylated hGDF15.

GDF15-Conjugate Efficacy in Chow Fed Dogs: GDF15-Fatty Acid Conjugate

Study Goal:

To assess the effects of subcutaneous administration of 0.05 mg/kg of aGDF15-fatty acid moiety conjugate according to the invention or vehiclecontrol on food intake in an acute setting (6 hour) and over a 96 hourperiod in the Beagle dog. Plasma samples were collected at various timepoints throughout the 14 day post-dose period in order to evaluate thePK profile of this compound. Body weight was determined throughout thestudy.

Animals:

Baseline body weights and treatments

TABLE 2 Dog ID Weight (kg) Treatment 50 12.65 Vehicle 62 8.85 Vehicle 7710.15 Vehicle 67 8.85 GDF15 73 9.95 GDF15 75 12.25 GDF15Dosing Procedure:

Dosing of Vehicle or GDF15 was performed after baseline body weight andblood sample collection. The GDF15-fatty acid moiety conjugate wassupplied as a 0.97 mg/ml solution and was dosed by subcutaneousinjection without dilution at 0.05 mg/kg. An equivalent volume of 30mmol/I Sodium Acetate pH 4 Vehicle (52 μl/kg) was given to the vehicleanimals by subcutaneous injection.

Blood Collection:

Blood samples were collected from the cephalic or jugular vein (3 ml, intubes containing EDTA and the protease inhibitors Diprotin A andAprotinin) and were placed on ice until centrifugation at 3,000 rpm for20 min at 4° C. Plasma was distributed in aliquots and stored at −70° C.until analysis. The following time points were collected: 0, 6.75,24.75, 48.75, 72.75 and 96.75 hours. Additional samples were collectedon days 7, 10 and 14.

Food Intake Measurements:

Measurement of ad libitum food intake was begun 45 minutes after dosing.This food intake measurement consists of two phases: an acutemeasurement (0-6 hours) and a sub-chronic measurement (0-96 hours).

From 0-2 hours, the dogs were given 500 g regular chow (Hill's J/Ddiet). At 2 hours, the remaining food was removed, weighed and another500 g chow was offered for the 2-4 hour period. At 4 hours, theremaining food was removed, weighed and another 300 g chow was offeredfor the 4-6 hour period. At 6 hours, the remaining food was removed andweighed. A blood sample was collected at this time (6.75 hours). Thedogs were then offered 500 g chow overnight. On the mornings of Day 1-4,remaining food was removed, weighed and a blood sample was collectedfrom each animal. On days 1-3 the dogs were then offered 500 g chow fora 24 hour period. On day 4, the dogs were returned to their normalallotment of chow (260 g).Additional Food Intake Measurements:

On days 7, 14 and 28 the study animals were given 6 hours to consumetheir daily chow (260 g). At the end of this time period, any remainingfood was collected and weighed.

Body Weight Measurements:

Body weights were measured at baseline and days 2, 4, 7, 10, 14, 18 and28. Baseline body weights were collected in the fasting state. Bodyweights collected on days 2 and 4 were not fasted. For the vehicletreated animals, all other body weights were collected in the fastedstate. For the GDF15 treated animals, the body weights determined ondays 7-28 were not fasted since the animals were given food continuouslyin order to stimulate appetite and regain weight.

Efficacy of GDF15-Fatty Acid Assay Conjugate of the Invention in ChowFed Dogs

Study Goal:

To assess the effects of subcutaneous administration of vehicle controland 0.015 mg/kg or 0.005 mg/kg GDF15-fatty acid moiety conjugate of theinvention on food intake in an acute setting (6 hour) and over a 96 hourperiod in the Beagle dog (In this study the vehicle arm will beperformed prior to the treatment arm in all dogs). Plasma samples willbe collected at various time points throughout the 14 day post-doseperiod in order to evaluate the PK profile of this compound. Body weightwas determined throughout the study.

Animals:

Baseline body weights and treatments

TABLE 3 Dog ID Weight (kg) Treatment Weight (kg) Treatment 29 12.75Vehicle 12.85  5 μg/kg hGDF15 57 13.35 Vehicle 13.80  5 μg/kg hGDF15 619.30 Vehicle 9.45  5 μg/kg hGDF15 77 10.70 Vehicle 11.15  5 μg/kg hGDF1545 11.90 Vehicle 12.20 15 μg/kg hGDF15 50 13.00 Vehicle 13.05 15 μg/kghGDF15 59 14.20 Vehicle 14.65 15 μg/kg hGDF15 72 8.80 Vehicle 9.05 15μg/kg hGDF15Dosing Procedure:

Dosing of Vehicle was performed after baseline body weight and bloodsample collection. 52 μl/kg of 30 mmol/l Sodium Acetate pH 4 Vehicle (52μl/kg) was given to the vehicle animals by subcutaneous injection.Dosing of GDF15 was performed after baseline body weight and bloodsample collection. The GDF15-fatty acid moiety conjugate was supplied asa 1.20 mg/ml solution and was dosed by subcutaneous injection afterdilution at 0.015 mg/kg and 0.005 mg/kg. The GDF15 stock was diluted inorder to maintain the 52 μl/kg delivered in a prior study.

Blood Collection:

Blood samples were collected for the vehicle and treatment arms of thestudy. Samples were collected from the cephalic or jugular vein (3 ml,in tubes containing EDTA and the protease inhibitors Diprotin A andAprotinin) and were placed on ice until centrifugation at 3,000 rpm for20 min at 4° C. Plasma was distributed in aliquots and stored at −70° C.until analysis. The following time points were collected: 0, 6.75,24.75, 48.75, 72.75 and 96.75 hours. Additional samples were collectedon days 7, 10 and 14.

Food Intake Measurements:

Food Intake was measured during both the vehicle and treatment arms ofthe study. Measurement of ad libitum food intake was begun 45 minutesafter dosing. This food intake measurement consists of two phases: anacute measurement (0-6 hours) and a sub-chronic measurement (0-96hours).

From 0-2 hours, the dogs were given 500 g regular chow (Hill's J/Ddiet). At 2 hours, the remaining food was removed, weighed and another500 g chow was offered for the 2-4 hour period. At 4 hours, theremaining food was removed, weighed and another 300 g chow was offeredfor the 4-6 hour period. At 6 hours, the remaining food was removed andweighed. A blood sample was collected at this time (6.75 hours). Thedogs were then offered 500 g chow overnight. On the mornings of Day 1-4,remaining food was removed, weighed and a blood sample was collectedfrom each animal. On days 1-3 the dogs were then offered 500 g chow fora 24 hour period. On day 4, the dogs were returned to their normalallotment of chow (260 g).

Additional Food Intake Measurements:

On various days between days 7 and 14 in the vehicle arm and betweendays 7 and 28 in the treatment arm, the study animals were given 6 hoursto consume their daily chow (225 g). At the end of this time period, anyremaining food was collected and weighed. Once a week, a timedmeasurement of food consumption was taken. Consumption of 225 g chow wasmeasured at 1, 2, 4 and 6 hours after feeding to determine whether eachdog's feeding pattern had returned to normal.

Body Weight Measurements:

Body Weight was measured during both the vehicle and treatment arms ofthe study. During the vehicle arm, body weights were measured atbaseline and days 2, 4, 7, 10 and 14. During the treatment arm, bodyweights were measured at baseline and days 2, 4, 7, 10, 14, 17, 21, 24and 28. Body weights collected on days 2 and 4 were not fasted. Allother body weights were determined in the fasting state.

Conjugate of Example 2 was Tested in Above Assay

Dog Single Sc Dose

TABLE 4 Body Duration of action weight Food Body change Food intakeIntake weight (%) change (FI) (BW) Dose (at 14 (% of vehicle) reductionreduction (ug/kg) days) (0-6 hrs) (0-96 hrs) (days) (days)  5 −5 55 45 714 15 −5 60 38 9 14 50 −13 50 26 7 18 dGDF15 — 31 (50 ug/kg)GDF15-Conjugate Improves Measures of Metabolic Disease IncludingDiabetes and Fatty Liver disease in obese mice

Diet-induced obese mice were dosed once weekly with vehicle or Example19Bm (0.5 mg/kg/s.c.) for 4 weeks. Non-fasted glucose and insulin weremeasured 2 weeks after the first dose, and overnight fasted bloodglucose and insulin were measured 4 weeks after the first dose. Example19Bm reduced non-fasted glucose by 23% (207.1 mg/dl vehicle treated vs.160.4 mg/dl Example 19Bm; p<0.05). Example 19Bm reduced non-fastedinsulin levels by 75% compared to vehicle treated mice (2.1 vs 8.7ng/ml; p<0.05). Four weeks after the initial dose, Example 19Bm reducedfasting blood glucose by 28% (142.7 vs. 199.5 mg/dl; p<0.05) and fastinginsulin by 78% (0.77 vs. 3.5 ng/ml; p<0.05). Markers of fatty liverdisease were also improved by four, once-weekly doses of Example 19Bm.Example 19Bm reduced hepatic steatosis by 57.5% (11.36 vs. 26.73% liverfat; p<0.05) and serum levels of a marker of hepatocyte damage, alanineaminotransferase (ALT), by 58% (46.2 vs. 110.5 U/L; p<0.05). Inaddition, Example 19Bm decreases the hepatic expression of PNPLA3, acausative gene in progressive liver diseases, by 77% (p<0.05).

The activity and plasma stability of the APJ-agonist conjugates ofExamples 20 and 21 according to the present invention can be assessed bythe following in vitro and in vivo methods described below.

hAPJ Calcium Flux Assay:

Chem-5 APJ stable cells (Millipore # HTS068C) were plated in 384-wellformat with 10,000 cells/well in 25 ul growth media, then grown 24 hoursin a 37° C. tissue culture incubator. One hour before the assay, 25ul/well FLIPR Calcium 4 dye (Molecular Devices R8142) with 2.5 mMprobenecid was added, and cells were incubated one hour in a 37° C.tissue culture incubator. Peptides were solubilized in HBSS, HEPES &0.1% BSA buffer, and serially-diluted 10-fold, from 50 uM to 5 pM, intriplicate. FLIPR Tetra was used to add peptide to the cells with dye(1:5, for final peptide concentrations ranging from 10 uM to 1 pM).FLIPR dye inside the cells emitted fluorescence after binding tocalcium, while fluorescence from outside the cells was masked.Fluorescence was measured using 470-495 excitation and 515-575 emissionwavelengths on the FLIPR Tetra. Readings were done for 3 minutes total,beginning 10 seconds before the peptide addition. Maximum-minimum valueswere calculated and plotted for each peptide concentration, and GraphPadprism software was used to calculate EC₅₀ values at the curve inflectionpoints, for calcium flux stimulation by peptides.

In Vivo Assay:

Conjugate was dissolved in PBS (Phosphate buffered saline) to aconcentration of 1 mg/ml to form Dosing solution. Dosing solution wasadministered intravenously to male Sprague-Dawley rats via lateral tailvein at a volume of 1 ml/kg body weight, corresponding to a dose of 1mg/kg. Venous blood samples were acquired from a jugular vein catheterat prescribed times after dosing and immediately placed on wet ice.These samples were centrifuged at 4C, with supernatant plasmatransferred to a fresh tube for analysis.

Bioanalysis:

-   -   Standard curve preparation: Stock solution was prepared by        dissolving peptide conjugate in water to 1 mg/ml. 10 uL of Stock        was mixed with 990 uL rat plasma to form a working stock of        10,000 ng/ml in plasma. This was serially diluted in plasma to        form standards of 5000, 1000, 500, 100, 50, 10, 5 and 1 ng/ml.    -   Sample and standard preparation: 25 uL plasma sample or standard        was transferred to a clean plate. 150 uL acetonitrile:MeOH (1:1)        containing 100 ng/ml glyburide as internal standard was added to        each vial and the plate vortexed to mix the contents. The plate        was centrifuged at 4000 rpm at 4C. 125 uL supernatant was        transferred to a clean plate, mixed with 50 uL water and        analyzed by LC/MS.        LC/MS Analysis:

HPLC: Agilent 1290 HPLC with autosampler

Column: MAC-MOD ACE C18, 3 μm, 30 mm×2.1 mm i.d.

Mobile phase A: 0.1% Formic acid in acetonitrile

Mobile phase B: 0.1% Formic acid in water

Gradient Program:

Time Flow Mobile Mobile (min) (mL) Phase A (%) Phase B (%) 0 0.7 98 20.5 0.7 98 2 1.5 0.7 5 95 2.5 0.7 5 95 2.6 0.7 98 2 3.1 0.7 98 2

Mass spectrometer: AB Sciex 6500

MS conditions: Q1 (m/z+) 809.3; Q3 (m/z+) 923.7; DP: 60; CE: 25

Data Analysis:

MS data were captured and analyzed using WatsonLIMS v7.4 software.

Activity and Stability of APJ Agonist-Conjugate of the Invention UsingAssays Described Supra

TABLE 5 hAPJ Ca²⁺ In vivo Plasma Flux EC₅₀ stability Peptide [nM] t½ [h]pE-R-P-C*-L-S-C*-K- 3 0.9 G-P-(D-Nle)- NH(Phenethyl) (disulfide C⁴-C⁷)(SEQ ID. NO: 28) pE-R-P-R-L-C*-H-K- 1.04 0.7 G-P-Nle-C*-F- OH(DisulfideC⁶-C¹²) (SEQ ID. NO: 29) Example 20A 2479 — Example 21A 65 7.4 Example21B 839 —

The activity and plasma stability of the oxytocin conjugates of Example26A and 26B according to the present invention can be assessed by thefollowing in vitro and in vivo methods described below.

In Vitro Assay Description:

Materials & Methods

Compound Plate Preparation

Supplied compounds were prepared in DMSO and ultimately prepared in theEurofins Discovery Services GPCRProfiler® Assay Buffer to concentrationsthat were three-fold higher than the final assay concentration.Similarly, vehicle controls and positive controls were prepared toensure all assays were properly controlled.Reference Controls

GPCR Reference Target Agonist Emax OT Oxytocin 1.25 μMAll wells were prepared using the Eurofins Discovery ServicesGPCRProfiler® Assay Buffer. The GPCRProfiler® Assay Buffer was amodified Hanks Balanced Salt Solution (HBSS) where HBSS was supplementedto contain 20 mM HEPES and 2.5 mM Probenecid at pH7.4.Calcium Flux AssayAgonist AssayCompound(s) supplied were plated in duplicate for each concentrationassayed.Reference agonist, oxytocin, was prepared in a similar manner to serveas assay control. The reference agonist, oxytocin, was included at Emax(the concentration where the reference agonist elicited a maximalresponse).The agonist assay was conducted on a FLIPRTETRA instrument where thetest compound(s), vehicle controls, and reference agonist were added tothe assay plate after a fluorescence/luminescence baseline wasestablished. The agonist assay was a total of 180 seconds and was usedto assess each compound's ability to activate each GPCR assayed.Upon completion of the three minute agonist assay, the assay plate wasincubated at 25° C. for a further seven (7) minutes.Data ProcessingAll plates were subjected to appropriate baseline corrections. Oncebaseline corrections were processed, maximum fluorescence/luminescencevalues were exported and data manipulated to calculate percentageactivation and percentage inhibition. Negative values of 0 to −30% maybe the result of biological variance. Data manipulation calculation isas followed: ((Max RLU)−(Baseline Avg.))/((Positive Avg.)−(BaselineAvg.))In Vivo Assay Description:Conjugate was dissolved in PBS (Phosphate buffered saline) to aconcentration of 3 mg/ml to form Dosing solution. Dosing solution wasadministered intravenously to male Sprague-Dawley rats via lateral tailvein at a volume of 1 ml/kg body weight, corresponding to a dose of 3mg/kg. Venous blood samples were acquired from a jugular vein catheterat prescribed times after dosing and immediately placed on wet ice.These samples were centrifuged at 4C, with supernatant plasmatransferred to a fresh tube for analysis.Bioanalysis:Standard curve preparation: Stock solution was prepared by dissolvingpeptide conjugate and peptide into two separate vials indimethylsulfoxide to 1 mg/ml. 10 uL of each stock was mixed with 980 uLrat plasma to form a working stock of 10,000 ng/ml in plasma. This wasserially diluted in plasma to form standards of 5000, 1000, 500, 100,50, 10, 5, 1, 0.5, and 0.1 ng/ml. Sample and standard preparation: 25 uLplasma sample or standard was transferred to a clean plate. 150 uLacetonitrile containing 100 ng/ml glyburide as internal standard wasadded to each vial and the plate vortexed to mix the contents. The platewas centrifuged at 4000 rpm at 4C. 125 uL supernatant was transferred toa clean plate, mixed with 150 uL water and analyzed by LC/MS.LC/MS Analysis:HPLC: Agilent 1290 HPLC with autosamplerColumn: MAC-MOD ACE C18, 3 μm, 30 mm×2.1 mm i.d.Mobile phase A: 0.1% Formic acid in acetonitrileMobile phase B: 0.1% Formic acid in waterGradient Program:

Time Flow Mobile Mobile (min) (mL) Phase A (%) Phase B (%) 0 0.7 98 20.5 0.7 98 2 2.0 0.7 2 98 2.5 0.7 2 98 2.6 0.7 98 2 3.0 0.7 98 2Mass spectrometer: AB Sciex 6500Peptide MS conditions: Q1 (m/z+) 945.20; Q3 (m/z+) 687.27; DP: 140; CE:39Peptide Conjugate MS Conditions: Q1 (m/z+) 1270.85; Q3 (m/z+) 468.30;DP: 140; CE: 77Data analysis: MS data were captured and analyzed using WatsonLIMS v7.4software.Activity and Stability of Oxytocin Fatty Acid Conjugate of the InventionAccording to Assays Described Supra

TABLE 6 In vivo Plasma OT Ca²⁺ Flux stability Peptide EC₅₀ [nM] t½ [h]Example 26A 8.4 46 Unconjugated 7.8 0.6 oxytocin analog Example 13 ofWO2014/095773Example 13 of WO2014/095773 is represented below:

The oxytocin-fatty acid conjugate of the invention demonstrates a 75fold increase in half-life compared to unconjugated oxytocin analog.

The activity and plasma stability of the AgRPconjugates of Example 27Aand 27 B according to the present invention can be assessed by thefollowing in vitro and in vivo methods described below.

A) HTRF cAMP Assay Protocol:

Passage of HEK293/MC4R Cells

-   -   Cell: HEK293/MC4R stable cell line    -   Complete medium: DMEM/F12 1:1 (Gibco, Cat. No. 11039, For assay,        no-phenol red medium Cat. No. 21041)        -   10% FBS (Heat inactivated, Gibco, Cat. No. 10082)        -   200 μg/mL Geneticin (Gibco, Cat. No. 10131)        -   15 mM Hepes (GIBCO, Cat No. 15630)        -   2 mM L-glutamine (GIBCO, Cat No. 25030)    -   Flask: 150 cm² tissue culture treated flask (Corning, Cat. No.        430825).        -   Aspirate conditioned medium        -   Wash with 25 mL of DPBS (Gibco, Cat. No. 14190), then            aspirate it            -   FBS inhibits Trypsin-EDTA treatment.        -   Add 2.5 mL of 0.05% Trypsin-EDTA (Gibco, Cat. No. 25300)        -   Leave a few minutes, then tap the flask a few time to detach            cells        -   Add 25 mL of the complete medium to stop Trypsin-EDTA            treatment            -   Cell preparation for assays, no-phenol red complete                medium have to be used.        -   Pipetting softly a few times to resuspend clumping cells        -   Transfer the suspension into a 50 mL centrifuge tube        -   Spin down at 1200 rpm for 3 min        -   Aspirate supernatant        -   Disperse the cells by softly tapping the bottom        -   Add 5-10 mL of the complete medium, then resuspend by softly            pipetting            -   Cell preparation for assays, no-phenol red complete                medium have to be used.        -   Transfer 0.5 mL of the suspension into a sample vial for            Vi-cell        -   Count cell number by using a Vi-cell *Record cell density            and viability every time        -   Transfer 1-3×10⁶ cells into a new 150 cm flask            -   For 3 days: 3×10⁶ cells/flask            -   For 4 days: 1×10⁶ cells/flask        -   Incubate at 37 C with 5% CO₂            Cell Seeding for HTRF cAMP Assay (One Day Before Assay)    -   Prepare cell suspension as in the passage section    -   Dilute the suspension to 2.34×10⁵ cells/mL        -   13 mL is enough for one 384 well plate.    -   Dispense 30 μL of the cell suspension into each well of a        Poly-D-Lysine BIOCOAT 384-well clear plate (Becton Dickinson,        Cat. No. 354660): 7000 cells/well        -   Poly-D-lysine coated plate is essential in this assay.        -   No cell for wells of cAMP standard    -   Incubate at 37 C with 5% CO₂ over night        HTRF cAMP Assay        1. Preparation of Reagents

1M IBMX

IBMX (MW 222.25 g/mol, ACROS Cat. No. 228420010) 111 mg DMSO (SigmaAldrich, Cat. No. D2650) 500 uL Store at 4° C.

40 mg/mL BSA Solution

Bovine serum albumin (Sigma A7030-50G) 200 mg dH₂O  5 mL Store at 4° C.

1 mg/mL (176 uM) AgRP Master Solution (in HBSS/2 mg/mL BSA)

R&D human AgRP C-terminal (Cat. No. 3726-AG-100) 100 ug/vial 1× HanksBuffered Salt Solution (HBSS)  95 uL (Gibco, Cat. No. 14065, w/Ca andMg) 40 mg/mL BSA solution  5 uL Store at 4° C.

2 mM NDP-aMSH Master Solution

NDP-aMSH (MW 1646.9, Bachem, Cat. No. H1100)  1 mg/vial dH₂O 304 uL

-   -   Once dissolved, dispense 10 uL aliquots into 200 uL tubes, then        store at −20 C

Assay Buffer 1

HBSS  10 mL 1M Hepes (Gibco, Cat. No. 15630) 0.2 mL 1M IBMX  20 uL

-   -   To avoid precipitation of IBMX, please vortex the buffer until        fully dissolved.

Assay Buffer 2

HBSS   20 mL 1M Hepes (Gibco, Cat. No. 15630)  0.4 mL 1M IBMX   40 uL 40mg/mL BSA solution 0.25 mL

-   -   To avoid precipitation of IBMX, please vortex the buffer until        fully dissolved.

6 nM NDP-aMSH for IgG Titration and AgRP Titration

2 uM NDP-aMSH (1000-fold dilution of the master solution)  10.8 uL Assaybuffer 1 3600 uL

-   -   Example for one 384 well assay

120 nM AgRP for IgG Titration

10-fold diluted master solution (17.6 uM)  26 uL Assay buffer2 3800 uL

-   -   Example for one 384-well plate

NDP-aMSH working solutions for titration (see reagents)

AgRP working solutions for titration (see reagents)

IgG working solutions for titration (see reagents)

cAMP standard solutions (see reagents)

2. Assay (2 Step Protocol)

Assay Kit: Cisbio cAMP HiRange HTRF Kit (Cat. No. 62AM6PEB)

-   -   Preparation of IgG/AgRP mix (1:1)        -   Mix 15 uL of IgG working solutions and 15 uL of 120 nM AgRP,            then incubate for 1 hr at ambient temperature    -   Preparation of assay plate        -   Discard culture medium by inverting the 384-well assay plate            containing cells on a Wipeall, then tapping in order to            remove the culture media.        -   Add 100 μL of DPBS to each well and discard in the same            manner            -   Once discard PBS, move the next as soon as possible to                avoid dry-up        -   Transfer 10 μL of the following reagents into each well            based on your sample alignment        -   cAMP standard: cAMP standards        -   Negative control for cAMP titration: Diluent in HTRF kit        -   Positive control: cAMP positive control in HTRF kit        -   MSH titration: Assay buffer 2        -   AgRP titration: AgRP working solutions        -   IgG titaration: IgG/AgRP mixture        -   Negative control for cell assay: Assay buffer 2    -   Flash spindown the 384 well plate at 1200 RPM    -   Incubated the cells for 15 minutes at an ambient temperature    -   Add 10 μL of the following reagents into each well based on your        sample alignment        -   cAMP standard: Assay buffer 1        -   Negative control for cAMP titration: Assay buffer 1        -   Positive control: Assay buffer 1        -   MSH titration: MSH working solutions        -   AgRP titration: 6 nM MSH solution        -   IgG titaration: 6 nM MSH solution        -   Negative control for cell assay: Assay buffer 1    -   Flash spindown the 384 well plate at 1200 RPM    -   Incubate the cells for an additional 30 minutes at an ambient        temperature        -   This incubation time is not so strict. +/−5 min should be OK            according to assay development data.    -   Add 10 μL of cAMP-d2 (diluted 1:4 in the lysis buffer provided        in the kit)        -   Important!! For Negative control, not cAMP-d2, but just the            lysis buffer    -   Add 10 μL of anti-cAMP Cryptate (diluted 1:4 in the lysis buffer        provided in the kit)    -   Flash spindown at 1200 RPM.    -   Incubate the assay plate for 45-60 min at an ambient        temperature.    -   Transfer 30 μL of each sample to a tissue culture treated white        polystyrene 384-well assay plate (Corning, Cat. No. 3572)    -   Flash spindown at 1200 RPM.    -   Measure the fluorescence with a Molecular device M5 or M5e with        the following setting.        Molecular Device M5/M5e Setting

Assay type Time-resolved fluorescence Integ delay 50 us Integration 400us Read Top read Wave length Ex 314 nm/Em668 nm Cutoff 630 nm Ex318nm/Em570 nm Cutoff 570 nm Auto mix Off Auto calibration On SensitivityReading 75 PMT On Plate 384 well standard oparque Setting time offColumn wavelength Column priority priority Carriage speed Normal Autoread Off

B) MC3 cAMP Assay

Materials:

Cells:

HEK293/MC3R stable cell line

Complete Medium:

-   -   DMEM/F12 1:1 (Gibco, Cat. No. 11039)    -   10% FBS (Heat inactivated, Gibco, Cat. No. 10082)×    -   200 μg/mL Geneticin (Gibco, Cat. No. 10131)    -   2 mM L-glutamine (GIBCO, Cat No. 25030)        Flask:

150 cm² tissue culture treated flask (Corning, Cat. No. 430825).

Assay Buffer

HBSS (Gibco—14175-095)  10 mL 1M Hepes (Fisher, Cat. No. BP299-1) 0.2 mL500 mM IBMX (MW 222.25 g/mol, ACROS  40 ul Cat. No. 228420010) BSA 0.25%Plates384 well solid bottom, Greiner bio-one (Cat no.—781080)Assay Protocol (Antagonist Protocol):

-   -   I. Aspirate conditioned medium    -   II. Wash with 2.5 mL of DPBS (Gibco, Cat. No. 14190)    -   III. Add 2 mL of 0.25% Trypsin-EDTA (Gibco, Cat. No. 25200-056)    -   IV. Leave the flask for few minutes in incubator, tap the flask        a few time to detach cells.    -   V. Add 10 mL of the complete medium to stop Trypsin-EDTA        treatment and mix it well by pipetting softly a few times to        re-suspend clumping cells    -   VI. Transfer 1.5 ml of cells into a new 150 cm flask containing        20 ml of complete media    -   VII. Transfer the remaining suspension into a 50 mL centrifuge        tube    -   VIII. Spin down at 1200 rpm for 4 mins. Aspirate supernatant    -   IX. Add 6 mL of the assay buffer to the tube and re-suspend the        cells by softly pipetting    -   X. Transfer 0.5 mL of the suspension into a sample vial for        Vi-cell and add another 0.5 ml of PBS.    -   XI. Count cell number by using a Vi-cell *Record cell density        and viability every time        -   i. Plate cells at 4K/well in 10 ul/well of assay buffer            containing IBMX.        -   ii. Leave the plate in incubator for ˜30 mins before assay            is started on suspension cells.            Two step cAMP protocol is followed for cAMP determination.            Procedure    -   I. To 10 ul/well of cells add 5 ul of AgRP prepared at 3× in        assay buffer only to antagonist wells.    -   II. Add 5 ul of buffer to positive control wells (wells that        will have NDP-α-MSH).    -   III. Incubate the plate at 37° C. for ˜20 mins.    -   IV. Add 5 ul/well of agonist EC80 (NDP-α-MSH) prepared at 4× to        wells containing AgRP DRC.    -   V. Add 5 ul/well of agonist (NDP-α-MSH) DRC prepared at 4×        (final highest concentration in plate is 100 nM) for NDP-α-MSH        EC50 calculation    -   VI. Add buffer only to negative control.    -   VII. Pulse spin the 384 well plate and incubate the cells for 30        minutes in incubator.    -   VIII. Add 10 μL of the following reagents into each well:        -   a. 10 μL of cAMP-d2        -   b. *Important!! For Negative control, do not add cAMP-d2,            but just the lysis buffer and 10 ul/well of Tb-cryptate        -   c. 10 μL of anti-cAMP Cryptate        -   d. Pulse spin the plate and incubate for 60 mins at room            temperature.

C. In Vivo Assay Description:

10 nanomoles of conjugate was dissolved in 300 μL of PBS (Phosphatebuffered saline) to form Dosing solution. Dosing solution (300 M) wasadministered intravenously to male Sprague-Dawley rats via lateral tailvein (corresponding to a dose 10 nanomoles per rat). Blood was collectedvia tail snip at prescribed times after dosing and immediately placed onwet ice. These samples were centrifuged at 4C, with supernatant plasmatransferred to a fresh tube for analysis.Bioanalysis:Standard Curve Preparation:

The two fatty acid conjugates of examples 27A and 27B and one maturehuman AgRP peptide were used to make standards. Intermediate stocksolutions of each AgRP were prepared by diluting the stock labeledpeptides in ELISA sample diluent with casein to 100 ug/ml. For assay,intermediates were diluted to a top standard concentration of 2500 pg/mLand then diluted 2-fold serially to 16 points including a zero dosestandard in ELISA sample diluent with bovine serum albumin (BSA).

Sample Dilution:

Plasma samples were diluted 10-fold and then 5-fold serially out to31,250-fold in ELISA sample diluent with BSA.

5B1 Human AgRP ELISA Method:

384 well microplates were coated with anti-human AgRP clone 5B1overnight at 30 uL/well in 1×PBS at room temperature (RT). Plates wereaspirated and blocked with a milk-based blocker at 90 uL/well for 2hours at RT. All further incubations were carried out at 30 uL/well.Plates were aspirated again and samples and standards were added to thewells for 2 hours at RT. Then the plates were washed three times with aphosphate based wash buffer with tween-20 and a biotinylated goatanti-human AgRP polyclonal antibody was added to the wells to detect thebound AgRP for 2 hours at RT. The plates were washed again and aHRP-labeled streptavidin reagent was added to the wells for 30 minutesat RT. Plates were washed a final time and a chemiluminescent substratewas added to all wells and plates were read immediately on a SpectramxM5 for light output.

Data Analysis:

Raw data was organized and analyzed for basic PK parameters.

Activity and Stability of AgRP Fatty Acid Conjugates of the InventionAccording to Assays Described Supra:

TABLE 7 In vivo Plasma MC4R EC50 MC3R EC50 stability Peptide [nM] [nM]t½ [h] Example 27A (mono 18 7 20 fatty acid conjugate) Example 27B (difatty 167 65 52 acid conjugate) AgRP 1.7 12 4.4

The activity and plasma stability of the FGF23 conjugate of example 28A,28B and 28C can be assessed by the following in vitro methods describedbelow.

In Vitro Activity Assay

Egr-1-Luciferase:

The biological activity of the purified hFGF23-FA conjugate was testedin Egr-1-luciferase reporter assays. Binding of the hFGF23-FA conjugateto the FGF23 receptor resulted in the downstream activation of Egr-1 andthe expression of a luciferase reporter regulated by the Egr-1 promoter.The Egr-1-luciferase reporter gene was constructed based on thatreported by Urakawa et al. (Nature, 2006, Vol 444, 770-774). HEK293Tcells seeded in 48-well poly-D-lysine plate were transfected with theEgr-1-luciferase reporter gene, the full-length transmembrane form ofKlotho and a transfection normalization reporter gene (Renillaluciferase). Five hours after the transfections, the transfection mixwas replaced with 3 ml DMEM plus 1% FBS containing graded concentrationsof the test protein. Cells were lysed 20 hours later in passive lysisbuffer (Promega, Cat #E194A) and luciferase activity was determinedusing Dual-Glo Luciferase Assay System (Promega, Cat #E2940).

Results

TABLE 8 Example EC50 (nM) Example 28B 1.195 Example 28C 0.258

The activity and plasma stability of the serelaxin fatty acid conjugatesof Examples 29A and 29B according to the present invention can beassessed by the following in vitro and in vivo methods described below.

In Vitro Activity Assays #1:

Materials:

DMEM: F12 media (Gibco, cat#11320)

IBMX (Sigma, cat#I5879)

384 solid bottom white plates (Greiner bio-one, cat #781945)

20,000 dynamic-2 cAMP kit (Cisbio, cat#62AM4PEC)

Adenosine 3′, 5′-cyclic monophosphate (Sigma, cat#A9501)

Matrix-plate mate plus (used for adding 5 μl of assays reagents)

PBS-Gibco (cat#10010-023)

Abbreviation Definition or Explanation cAMP cyclic adenosinemonophosphate RXFP1 Relaxin/Insulin related receptor DMSO dimethylsulfoxide HTRF Homogeneous Time Resolved Fluorescence 8k Eight thousandcAMP-d2 cAMP labeled with the dye d2 PDL Poly-d-lysine ul Microliter @at o/N Overnight uM Micromolar Min Minutes 37c 37 centigrade 3x Threetimes hr Hour DMEM: F12 Dulbecco's Modified Eagle Medium: NutrientMixture F-12 (DMEM/F-12) rhRLX Recombinant human relaxin RPMI RoswellPark Memorial Institute (RPMI) 1640 Medium FRET Fluorescence resonanceenergy transfer HEK293 Human embryonic kidney 293 cells IBMX3-Isobutyl-1-methylxanthine nM nano molar std Standard con ConcentrationPBS Phosphate buffer saline cpd compound HS Human serum HBSS Hanksbuffered saline solutionProtocol:Day 1: Seeded RXFP1-HEK293/parental HEK293 cells 8 k in 10 μl of DMEM:F12 media in solid bottom PDL coated white platesDay 2: Ran assay with the compoundAgonist Mode (Overview):

-   -   Cells in 10 μl of DMEM:F12 media @37° C. O/N    -   5 μl of 2000 μM(4×) of IBMX to the cells for 30 min at 37° C.    -   5 μl of 4× compound/Serelaxin to the above for 30 min at 37° C.        -   (from step 3 of cpd dilution, 400 nM—final is 100 nM top)    -   10 μl of cAMP-d2 conjugate    -   10 μl of Anti-cAMP cryptate conjugate    -   Incubate for 1 hr at RT    -   Read FRET signal—Envision 665 nm/620 nm        Compound Preparation        Serelaxin:    -   1) Diluted the stock 683.3 μM, i.e. 11.7 μl in 188.3 PBS pH7.4        diluted 3× times in PBS by transferring 15 μl of cpd to 30 μl        (final is 40 μM)—by Hand    -   2) Diluted 1:10, i.e 6 μl of above in 54 μl of DMEM:F12 (final        is 4 μM)—by Hand    -   3) Diluted 1:10, i.e 10 μl of above in 90 μl of DMEM:F12 (final        is 400 nM)—by Hand        Serelaxin—FA Conjugate    -   1) Diluted the stock to 40 μM in PBS pH7.4 diluted 3× times in        PBS by transferring 15 μl of compound to 30 μl    -   2) Diluted 1:10, i.e 6 μl of above in 54 μl of DMEM:F12—by Hand    -   3) Diluted 1:10, i.e 10 μl of above in 90 μl of DMEM:F12 (1:100        dilution)—by Hand        Fatty Acid    -   1) Diluted the stock to 40 μM in PBS pH7.4 diluted 3× times in        PBS by transferring 15 μl of cpd to 30 μl    -   2) Diluted 1:10, i.e 6 μl of above in 54 μl of DMEM:F12—by Hand    -   3) Diluted 1:10, i.e 10 μl of above in 90 μl of DMEM:F12 (1:100        dilution)—by Hand        cAMP Standard Curve Dilutions:    -   1. 150 μl of the cAMP std diluted in DMEM:F12 media to first        column (2800 nM)    -   2. 100 μl of the DMEM:F12 media to the subsequent columns till        10 (1-11)    -   3. 3× dilutions, by transferring 50 μl to 100 μl    -   4. 20 μl from the step 3 to appropriate wells of the std curve        plate    -   5. 10 μl of Anti-d2 & Anti-cAMP cryptate conjugate    -   6. Incubate 1 h at room temperature    -   7. Read FRET signal—Envision 665 nm/620 nm        Analysis:        The cAMP nM concentration was Log (x) transformed using Graph        pad prism        The cAMP amount was interpolated from the standard curve using 4        parameter nonlinear regression.        The interpolated values were converted to nM using 10{circumflex        over ( )}Y transformation        The computed cAMP amounts were plotted against the compound        concentration, using 4 parameter nonlinear regression        Results:

TABLE 9 Compound EC50 (nM) Serelaxin 1.12 Serelaxin-FA conjugate Example29B 4.51In Vitro Activity in Presence of Bovine Serum Albumin and Human Serum#2:Materials:DMEM: F12 media (Gibco, cat#11320)IBMX (Sigma, cat#15879)384 solid bottom white plates (Greiner bio-one, cat #781945)20,000 dynamic-2 cAMP kit (Cisbio, cat#62AM4PEC)Adenosine 3′, 5′-cyclic monophosphate (Sigma, cat#A9501)Matrix-plate mate plus (used for adding 5 μl of assays reagents)PBS-Gibco (cat#10010-023)1M HEPES Gibco (cat-15630-080)1×HBSS Gibco (cat-14175-095)Assay buffer—1×HBSS+10 mM HEPESBovine serum albumin cat# A2153 (Sigma-Aldrich)Sigma Aldrich—H4522 (Human serum)Conditions:

-   -   600 μM of BSA    -   4% Human Serum    -   10% Human Serum (Sigma aldrich—H4522)    -   Assay buffer        Compounds Tested:    -   Serelaxin    -   Serelaxin-FA (Example 29A)        Compound Handling:        Serelaxin-:

Stock is (796.57 uM) i.e 4.75 mg/ml MW is 5963 Daltons

-   -   Serelaxin 10 ul stock dissolved in 190 ul of PBS, final        concentration is 40 μM, which are diluted 3× fold by        transferring 30 ul to 60 ul of the assay buffer 11 point curve,        (A2-A12) 12^(th) is zero.

Serelaxin-FA Conjugate:

Stock is (287.79 uM) i.e 2.61 mg/ml MW is 9069 Daltons

-   -   Serelaxin 27.798 ul stock dissolved in 172.2 ul of PBS, final        concentration is 40 μM, which are diluted 3× fold by        transferring 30 ul to 60 ul of the assay buffer 11 point curve,        (A2-A12)₁₂th is zero.        BSA Stock Solution Preparation:        For 666.66 uM BSA: Made assay buffer 30 mls by dissolving 1.32        gms of BSA        For 600 uM BSA: Made assay buffer 30 mls by dissolving 1.18 gms        of BSA        No BSA, has only assay buffer        Human Serum        For 4.44% HS: 1.34 mls in 28.66 mls of assay buffer        For 4% HS: 1.2 mls in 28.8 mls of assay buffer        For 11.11% HS: 3.33 mls in 26.67 mls of assay buffer        For 10% HS: 3 mls in 27 mls of assay buffer        Procedure:        Day 1: Seed 8,000 cells/well of RXFP1-HEK293 and HEK293        (parental) cells in 10 μl/well volume in basal DMEM:F12 media-on        solid bottom plate. Incubate cells overnight at 37° C./5% CO₂.        Day2:    -   1. Wash cells 2× times with 50 ul of assay buffer and were        tapped gently on paper towel to get rid of assay buffer after        first and second wash    -   2. Cells were pretreated with 15 ul of media with IBMX (666.66        uM) containing respective media (600 uM of BSA, 4% BSA, 10%        Human serum and assay buffer alone) for 30 minutes at 37° C.    -   3. Serially dilute cpds 3× times, 11 point curve—transferring 15        ul of cpds from previous well to subsequent well with 30 ul of        PBS, well 11 is PBS only    -   4. Dilute (1:10) in assay buffer from step 3 (i.e is 10 ul to 90        ul of assay buffer)    -   5. Dilute again from step 4 (1:10) in respective media (666.66        μM of BSA &4.44% & 11.11% Human serum and assay buffer) final        con of the BSA is 600 μM and Human serum is 4 & 10%    -   6. (*Incubate the cpds for 1 hr at RT in their respective media,        before adding to cells)    -   7. 5 ul of from step 6 i.e is 4× Serelaxin/Serelaxin-FA to 15 ul        of cells for 30 more minutes at 37° C. (top concentration of        Serelaxin is 100 nM)    -   8. Add 10 ul cAMP d2 conjugate    -   9. Add 10 ul anti-cAMP-Cryptate    -   10. Incubate for 1 hr at room temperature    -   11. Read FRET on Envision    -   12. cAMP std curves were made in their respective media.        cAMP Standard Curve Dilutions:        The initial stock of cAMP standard is 1120000 nM    -   1. Dilute the initial stock (1:4) by dissolving 20 ul of the        cAMP stock in 60 ul of assay buffer    -   2. (1:10) dilution of step 1 in assay buffer (i.e is 20 ul in        180 ul of assay buffer)    -   3. (1:10) dilution of step 2 in respective concentration of        4.44% & 11.11% HS, 666.66 uM BSA or No BSA—The final        concentration would end up to be 4%, 10% of HS and 600 μM of        BSA.        cAMP Standard Curve    -   1. Add 150 μl of the respective cAMP standards to first column        (2800 nM)    -   2. Add 100 μl of the assay buffer with respective concentrations        600 uM BSA, 4% &10% HS and 0%) to the subsequent 11 columns i.e        is (2-12)    -   3. 3× dilutions, by transferring 50 μl to 100 μl of subsequent        wells 12^(th) well is Zero no cAMP    -   4. 20 μl from the step 3 to appropriate wells of the std curve        plate    -   5. Add 10 μl of d2 conjugate    -   6. Incubate 1 hr at room temperature    -   7. Read on HTRF-Envision        Analysis:        The cAMP nM concentration is Log (x) transformed using Graph pad        prism        The cAMP amount was interpolated from the standard curve using 4        parameter nonlinear regression.        The interpolated values were converted to nM using 10{circumflex        over ( )}Y transformation        The computed cAMP amounts were plotted against the compound        concentration, using 4 parameter nonlinear regression        Result:

TABLE 10 Ec₅₀ (nM) 0% 4% 10% 600 uL Plasma Plasma Plasma BSA SeRelaxin 80.3 0.4 0.7 FA-SeRelaxin (Ex 29a) 100 11 15 15In Vivo Assay:Compounds (serelaxin and serelaxin conjugates) can be tested in variousrodent models to evaluate short- and long-term cardiovascular responses.Short-Term Models—

Mice (any strain, but DBA/2 preferred) or rats (any strain, butSprague-Dawley preferred) are anesthetized with inhaled isoflurane,maintained at a stable surgical plane of anesthesia with ˜2% isofluranein 100% oxygen, and rectal temperature maintained at a normal level. Acarotid artery and jugular vein (mice) or a femoral artery and vein(rat) are exposed through overlying skin incisions, and the vesselscatheterized. The arterial catheter is connected to a pressuretransducer and the signal is directed to a digital data acquisitionsystem (e.g., Ponemah) for continuous measurement of arterial pressureand triggering of heart rate. Alternatively, heart rate is triggered byan electrocardiogram signal recorded via subcutaneously inserted needleelectrodes. After allowing the arterial pressure and heart rate tostabilize, a cocktail of autonomic blocking agents (e.g., atropine andpropranolol at 2 mg/kg each) are administered intravenously over ˜3-4minutes. When cardiovascular parameters re-stabilize, serelaxin orserelaxin conjugates are injected as an intravenous bolus over ˜3 sec.Relaxin elicits an increase in heart rate with a characteristic slowonset (peak response in ˜6 minutes) and sustained duration of action(hours). In the same animal preparation, ventricular cardiac function(e.g., ejection fraction, fractional shortening, cardiac output) ismeasured by collecting serial echocardiographic images, which areanalyzed offline.

Long-Term Models—

Mice (any strain, but DBA/2 preferred) or rats (any strain, butSprague-Dawley preferred) are anesthetized with inhaled isoflurane andmaintained at a stable surgical plane of anesthesia with ˜2% isofluranein 100% oxygen. Analgesics are administered peri- and post-operatively.An artery and vein are cannulated as described above, but the cathetersare exteriorized through the dorsal skin region, flushed with heparinizesaline, and plugged with a stainless-steel pin. A subcutaneous cathetermight also be implanted subcutaneously in mice and exteriorized in asimilar fashion. In rats, the catheters are directed through aspring-tether/swivel system. On the day of the study, arterial cathetersare connected to pressure transducers, autonomic blockade is achieved asdescribed above except that the blocking agents can also be administeredvia the subcutaneous catheter in mice, and the blockade in both speciesis maintained thereafter by continuous intravenous or subcutaneousinfusions of the autonomic agents. Arterial pressure and heart rate aremonitored continuously with a digital data acquisition system. Afterallowing the arterial pressure and heart rate to stabilize, theautonomic blocking agents are administered intravenously orsubcutaneously over ˜3-4 minutes. When cardiovascular parametersre-stabilize, serelaxin or serelaxin conjugates are injected as anintravenous bolus over ˜3 sec. For assessing ventricular cardiacfunction and heart rate over a period of weeks in mice or rats,serelaxin conjugates are injected subcutaneously 1-3 times per week andserial echocardiographic images collected at baseline and weeklythereafter.

Serelaxin Source:

Serelaxin (Recombinant 1-chain human relaxin)

Connetics corporation, lot #00L605

1.0 mg/mL (5 mL vial) in 20 nM Na acetate buffer (pH 5.0)

Dilution of stock solution in vehicle to the desired concentration ofserelaxin for each dose.

The activity and plasma stability of the PIP conjugate of example 30 canbe assessed by the following in vitro and in vivo methods describedbelow.

Glucose-Stimulated Insulin Secretion (GSIS) Assay:

GSIS test was performed as a measurement of in vivo pancreatic beta cellfunction following recombinant human Prolactin-inducible Protein (hPIP)in high fat diet-induced obese (DIO) mice. Briefly, mice (m=5-7/group)were fasted overnight (5:00 PM-8:00 AM) and on the test day body weightand blood glucose (BG; determined with Embrace glucose meters) which wasdesignated as baseline timepoint. Next, mice were administered with hPIP(native and FA-conjugated PIP; solution in PBS; administered at 4 ml/kgbody weight) or a vehicle-control (PBS) once intravenuously (IV).Forty-five min following the hPIP administration, all mice were dosedwith oral glucose (3 g/kg dextrose; solution in PBS; administered at 4ml/kg body weight). Blood glucose was measured immediately before theglucose load (designated as 0 min time point) and at 15 and 30 minpost-glucose. Blood samples were collected for plasma isolation andmeasurement of plasma insulin were carried out at 0, 15 and 30 minpost-glucose.Pharmacokinetics (PK) Assay:Plasma exposure of hPIP were measured in DIO mice following a single IVadministration. Briefly, freely-fed DIO mice (n=2) were administeredwith hPIP (native and FA-conjugated PIP; solution in PBS; administeredat 4 ml/kg body weight) once intravenuously (IV). Blood samples werecollected and plasma isolated at 0.25, 0.5, 1, 3, 7, 24 and 48 hrspost-dose by and in-house ELISA assay (protocol shown below).Assay were Measured by the Following Steps:

-   -   Plates were coated overnight at room temperature with 30 ul hPIP        antibody (designated as PIP-8-AB; produced in-house; NBC        clone#87.19G9A11, at 8 ug/ml in PBS).    -   Aspirated before blocking 2 hr with 100 ul blocking reagent.    -   Aspirated and 30 ul samples added to incubate for 2 hrs, samples        and standards are diluted in Casein buffer (1% Casein, 1.7 mM        Sodium Phosphate Monobasic, 8.1 mM Sodium Phosphate Dibasic        Heptahydrate, 0.15M Sodium Chloride, 0.7% Triton X-100, and 0.1%        Sodium Azide.)    -   Plates were washed 3× 100 ul with Teknova wash buffer (0.05%        Tween in PBS)    -   30 ul biotinylated PIP antibody (designated as PIP-6 Ab;        produced in-house, MBC clone#87.8C6B3, at 10 ug/ml in casein        buffer) and incubates for 1 hr    -   Washed as above    -   Added 30 ul Streptavidine-HRP (Pierce cat #21140, at 0.4 ug/ml        in HRP buffer) HRP buffer (0.4% Casein, 1.7 mM Sodium Phosphate        Monobasic, 8.1 mM Sodium Phosphate Dibasic Heptahydrate, 0.15M        Sodium Chloride, and 0.1% Chloroacetamide) incubate 30 min.    -   Washed as above    -   Added 30 Femto Chemiluminescent Substrate (Thermo cat #34096)        and read immediately        Activity and Stability of PIP Fatty Acid Conjugate of the        Invention According to Assays Described Supra

TABLE 11 In vivo Plasma Insulin Plasma AUCB stability Cmax Peptide(ng/mL * min)** t½ [h] (nM) MRT (hr) Example 30 +75% ---13.8- 497.1 18.1Unconjugated PIP +29% --18.0- 219.8 12.0 **compared to the vehicleThe PIP fatty acid conjugate of the invention has an extended exposureresulting in an improved efficacy

The activity and plasma stability of the NPFF conjugate of example 31can be assessed by the following in vitro methods described below.

cAMP Assay Protocol with Cisbio cAMP Kit

The NPFF-FA conjugate of the invention was tested in presence ofForskolin in the assay described below

Reagents/Materials

Vendor Cat# Location (Stock) Greiner 384 clear bottom plate GreinerBio-One 781944 precoated with Poly-Lysine cAMP kit Cisbio 62AM4PEJ 4°C./−20° C. DMSO Sigma D2650 cAMP standard (1.12 mM in assay Sigma A9501−80° C. buffer + IBMX) Forskolin 5 mM Stock solution Sigma F6886 −20° C.(DMSO) IBMX 250 mM stock solution Sigma I5879 −20° C. (DMSO) HBSSInvitrogen 14175-095 R.T HEPES invitrogen 15630-080 R.T PTX (PertussisToxin) Sigma P2880 −20° C. BSA Free Fatty Acid (30%) Sigma A9205    4°C.Day 1: Plate Cells into 384-Well Plate.Following “subculturing protocol”

-   -   1. Make growth media without antibiotics (if necessary).    -   2. Equilibrate Growth medium without antibiotics bottle in        37° C. water bath after spraying with 70% ethanol.    -   3. Detach cells with Versene (3 ml per T.75 flask).    -   4. Transfer into 50 ml Falcon tube containing 17 ml of growth        media.    -   5. Centrifuge 4 min at 150 g.    -   6. Resuspend cell pellet in 10 ml Stimulation Buffer. Count        cells.    -   7. Prepare cell suspension in growth media with antibiotics at        5′000 cells/50 ul.    -   8. Plate 50 ul of cell suspension using the Viaflow 384-125 ul        pipet.    -   9. Let plates sit under the TC hood for 15 min.    -   10. Incubate at 37° C., 5% CO₂ and 90% humidity.        Day 2:        Preparation of Reagents Solutions:    -   1. Assay Buffer:    -   500 ml HBSS+10 ml HEPES. Store at R.T    -   Assay Buffer: 250 ml HBSS/HEPES+250 ul IBMX 1000× solution+0.1%        BSA (825 ul).    -   Make fresh daily.    -   2. Forskolin 2× Solution: 1 uM final in assay:        -   For cpds dilutions: 40 ul FSK/100 ml Assay buffer.        -   For NPFF dilution: 10 ul DMSO/10 ml 2×FSK.    -   3. NPFF dilutions: stock solution 1 mM in dH₂O— Final in assay 1        uM        -   a. 5 ul stock/625 ul ul FSK 2×/DMSO.        -   b. 100 ul sol. a +300 ul FSK 2×/DMSO. Final in assay 1 uM.        -   c. Make 1o dilution steps 1/4: 100 ul+300 ul in FSK 2×/DMSO.    -   4. Cpds dilutions: stock solution 10 mM in DMSO—Final in assay        40 uM        -   a. 5 ul stock/625 ul FSK 2×.        -   b. Make 11 dilution steps 1/4: 100 ul+300 ul FSK 2×.    -   5. cAMP standard:        -   a. 10 ul cAMP standard stock (1.12 mM)+90 ul Assay buffer.        -   b. 10 ul dilution a +90 ul stimulation buffer.        -   c. 20 ul dilution b+428 ul stimulation buffer: 500 nM.        -   d. Make 11 dilutions starting from dilution c: 100 ul            dilution c+100 ul stimulation buffer.    -   6. cAMP Detection Reagents:        -   a. d2-cAMP: 1000 ul/20 ml Lysis buffer. (250 ul/5 ml for one            384-well plate).        -   b. Cryptate conjugate: 1000 ul/20 ml Lysis Buffer. (250 ul/5            ml for one 384-well plate).            Assay Procedure            Step One:    -   1. Prepare Stimulation buffer.    -   2. Put Cisbio Lysis buffer at R.T.    -   3. Prepare the WellMate (wash with 70% alcohol followed by dH₂O        and HBSS/HEPES).    -   4. Prepare Forskolin and cpds dilutions.        Step Two: Stimulation with Forskolin    -   1. Wash cells with 50 ul of stimulation buffer:        -   a. Gently tap plates to remove O/N media.        -   b. Put white paper towel on top of plate and centrifuge            plate upside down for 20 sec at 300 RPM using the VWR            Symphony 4417 centrifuge.        -   c. Add 50 ul of stimulation buffer using the WellMate.        -   d. Gently tap plates to remove O/N media.        -   e. Put white paper towel on top of plate and centrifuge            plate upside down for 20 sec at 300 RPM using the VWR            Symphony 4417 centrifuge.        -   f. Check at cells under the microscope.    -   2. Add 10 ul of Stimulation buffer containing IBMX using Viaflow        384    -   3. Add 10 ul of 2× Forskolin solution containing the cpds using        the Viaflow 384 (on the 7^(th) floor). Mix solution before        adding to cells.    -   4. Incubate 30 min at R.T. (put plate in a drawer to avoid        temperatures changes).    -   5. Prepare cAMP standard curve and cAMP detection reagents.    -   6. Add 20 ul/well of cAMP standard curve to the standard curve        plate (same plate than assay plate).        Step Three: LANCE cAMP Assay    -   1. Add 10 ul d2 cAMP/well to assay plate using Combi 384.    -   2. Add 10 ul/well Cryptate conjugate to assay plate manually.    -   3. Seal plate.    -   4. Incubate 1 hour minimum at R.T (plate can read within 24        hour).    -   5. Put Tape on the bottom of the plate.    -   6. Read on Envision program “Cisbio 384 full plate”    -   7. See raw data into accessories files.    -   8. Data analyzed with GraphPad (see file in accessories files).        Results:

TABLE 12 Human R2 0.1% BSA (FFA) 3% BSA (FFA) 0.1% HSA 3% HSA compoundIC50 (nM) n = 4 IC50 (nM) n = 4 IC50 (nM) n = 2 IC50 (nM) n = 2 NPFF0.43 (+/− 0.11) 0.36 (+/− 0.17) 0.63 (+/− 0.25)  1.9 (+/− 1.7) Example31 53.9 (+/−26)  133 (+/−47) 33.5 (+/−3.11) 98.5 (+/−19) Human R1 0.1%BSA (FFA) 3% BSA (FFA) 0.1% HSA 3% HSA compound IC50 (nM) n = 4 IC50(nM) n = 4 IC50 (nM) n = 2 IC50 (nM) n = 2 NPFF 6.23 (+/− 3.3)  7.2 (+/−6.0)  8.4 (+/− 0.6) 33.4 (+/− 29) Example 31 >1000 >4000  620 (+/−142) >4 uM

The activity and plasma stability of the conjugate of the inventionwherein the biomolecule is a siRNA can be assessed by the following invivo methods described below.

Methods

Conjugate of Example 24 is a compound of the APOC3 siRNA conjugated withGalNAc (Reference Example 3) and the fatty acid. Human APOC3 transgenicmice (B6; CBA-Tg(APOC3)3707Bres/J) were purchased from the JacksonLaboratory (Bar Harbor, Me.). Mice were fed standard rodent chow andwater ad libitum with a 12 h light/dark cycle. Under this condition,APOC3 transgenic mice spontaneously develop hypertriglyceridemia withmarkedly increased plasma TG content (Aalto-Setala K, J Clin Invest1992). Four mice in each group were injected subcutaneously with eitherreference example 3 (APOC3 siRNA-GalNAc) or conjugate of Example 24 at adose of 25 mg/Kg of body weight. Blood was collected at baselineimmediately before injection, and 2, 4, 7 and 14 days after theinjection. Plasma was used to measure human APOC3 protein levels with anHTRF assay from Cisbio. One-way ANOVA was used to compare thestatistical difference between groups.ResultsBaseline plasma APOC3 levels were 176±21 mg/dL and 178±9 mg/dL in thereference example 3 and the conjugate 24 groups, respectively. Referenceexample 3 time-dependently decreased plasma APOC3 levels, by 56% fivedays after dosing versus baseline levels. By comparison, conjugate ofexample 24 decreased plasma APOC3 more effectively, with an 80% decreasefive days after dosing (FIG. 1). The duration of action is similar forboth reference example 3 and example 24 as shown in FIG. 1.

The conjugate of the present invention have plasma stability of at least5 h, at least 10 h, at least 20 h, at least 30 h, at least 40 h or atleast 50 h. In one embodiment, the plasma stability improvement comparedto the non-conjugated biomolecule is at least 2 fold, 5 fold, 10 fold,20 fold, 30 fold, 40 fold or 50 fold or 75 fold.

Combination Therapy

The conjugate of the present invention may be administered eithersimultaneously with, or before or after, one or more other therapeuticagent. The conjugate of the present invention may be administeredseparately, by the same or different route of administration, ortogether in the same pharmaceutical composition as the other agents.

In one embodiment, the invention provides a product comprising aconjugate of any one of preceeding embodiments or a mixture ofconjugates as described in embodiments 10 and 13, and at least one othertherapeutic agent as a combined preparation for simultaneous, separateor sequential use in therapy. In one embodiment, the therapy is thetreatment of a metabolic disorders or diseases, type 2 diabetesmellitus, obesity, dyslipidemia, elevated glucose levels, elevatedinsulin levels and diabetic nephropathy in a subject in need thereof,comprising: administering to the subject a therapeutically effectiveamount of a conjugate of the invention, or an amide, ester or saltthereof, wherein the biomolecule is human Growth Differentiation Factor15 (GDF15), homologs, variants, mutants, fragments and other modifiedforms thereof.

Products provided as a combined preparation include a compositioncomprising a conjugate of any one of the preceeding embodiments, and theother therapeutic agent(s) together in the same pharmaceuticalcomposition, or a conjugate of any one of the preceeding embodiments,and the other therapeutic agent(s) in separate form, e.g. in the form ofa kit.

In one embodiment, the invention provides a pharmaceutical compositioncomprising a conjugate of any one of the preceeding embodiments or amixture of conjugates according to embodiment 10 or 13, and anothertherapeutic agent(s). Optionally, the pharmaceutical composition maycomprise a pharmaceutically acceptable excipient, as described above.

In one embodiment, the invention provides a kit comprising two or moreseparate pharmaceutical compositions, at least one of which contains aconjugate according to any one of the preceeding embodiments. In oneembodiment, the kit comprises means for separately retaining saidcompositions, such as a container, divided bottle, or divided foilpacket. An example of such a kit is a blister pack, as typically usedfor the packaging of tablets, capsules and the like.

The kit of the invention may be used for administering different dosageforms, for example, oral, subcutaneous and parenteral, for administeringthe separate compositions at different dosage intervals, or fortitrating the separate compositions against one another. To assistcompliance, the kit of the invention typically comprises directions foradministration. In the combination therapies of the invention, theconjugate of the invention and the other therapeutic agent may bemanufactured and/or formulated by the same or different manufacturers.Moreover, the conjugate of the invention and the other therapeutic maybe brought together into a combination therapy: (i) prior to release ofthe combination product to physicians (e.g. in the case of a kitcomprising the conjugate of the invention and the other therapeuticagent); (ii) by the physician themselves (or under the guidance of thephysician) shortly before administration; (iii) in the patientthemselves, e.g. during sequential administration of a conjugate of theinvention and the other therapeutic agent.

The invention also provides the use of a conjugate according to any oneof preceeding embodiments, for treating a disease or condition set forthherein, wherein the patient has previously (e.g. within 24 hours) beentreated with another therapeutic agent. The invention also provides theuse of another therapeutic agent for treating a disease or condition setforth herein, wherein the patient has previously (e.g. within 24 hours)been treated with a conjugate according to any one of preceedingembodiments.

The term “in combination with” a second agent or treatment includesco-administration of the conjugate of the invention (e.g., a conjugateaccording to any one of the preceeding embodiments or a conjugateotherwise described herein) with the second agent or treatment,administration of the compound of the invention first, followed by thesecond agent or treatment and administration of the second agent ortreatment first, followed by the conjugate of the invention.

The terms “second agent” and “co-agent” are used interchangeably andinclude any agent which is known in the art to treat, prevent, or reducethe symptoms of a disease or disorder described herein, e.g. a disorderor disease selected from a metabolic disorders or diseases, type 2diabetes mellitus, obesity, pancreatitis, dyslipidemia, alcoholic andnonalcoholic fatty liver disease/steatohepatitis and other progressiveliver diseases, insulin resistance, hyperinsulinemia, glucoseintolerance, hyperglycemia, metabolic syndrome, hypertension,cardiovascular disease, atherosclerosis, peripheral arterial disease,stroke, heart failure, coronary heart disease, diabetic complications(including but not limited to chronic kidney disease), neuropathy,gastroparesis and other metabolic disorders.

In one embodiment, the therapy is the treatment of metabolic disordersor diseases, type 2 diabetes mellitus, obesity, pancreatitis,dyslipidemia, alcoholic and nonalcoholic fatty liverdisease/steatohepatitis and other progressive liver diseases, insulinresistance, hyperinsulinemia, glucose intolerance, hyperglycemia,metabolic syndrome, hypertension, cardiovascular disease,atherosclerosis, peripheral arterial disease, stroke, heart failure,coronary heart disease, diabetic complications (including but notlimited to chronic kidney disease), neuropathy, gastroparesis and othermetabolic disorders, in a subject in need thereof, comprising:administering to the subject a therapeutically effective amount of aconjugate of the invention, or an amide, ester or salt thereof, whereinthe biomolecule is human Growth Differentiation Factor 15 (GDF15),homologs, variants, mutants, fragments and other modified forms thereof.

Examples of second agents to combine with a conjugate of the instantinvention, wherein the biomolecule is human Growth DifferentiationFactor 15 (GDF15), homologs, variants, mutants, fragments and othermodified forms thereof; include:

1. Antidiabetic agents, such as insulin, insulin derivatives andmimetics; insulin secretagogues such as the sulfonylureas (e.g.,chlorpropamide, tolazamide, acetohexamide, tolbutamide, glyburide,glimepiride, glipizide); glyburide and Amaryl; insulinotropicsulfonylurea receptor ligands such as meglitinides, e.g. nateglinide andrepaglinide; thiazolidinediones (e.g., rosiglitazone (AVANDIA),troglitazone (REZULIN), pioglitazone (ACTOS), balaglitazone,rivoglitazone, netoglitazone, troglitazone, englitazone, ciglitazone,adaglitazone, darglitazone that enhance insulin action (e.g., by insulinsensitization), thus promoting glucose utilization in peripheraltissues; protein tyrosine phosphatase-1B (PTP-1B) inhibitors such asPTP-112; Cholesteryl ester transfer protein (CETP) inhibitors such astorcetrapib, GSK3 (glycogen synthase kinase-3) inhibitors such asSB-517955, SB-4195052, SB-216763, NN-57-05441 and NN-57-05445; RXRligands such as GW-0791 and AGN-194204; sodium-dependent glucosecotransporter inhibitors such as T-1095; glycogen phosphorylase Ainhibitors such as BAY R3401; biguanides such as metformin and otheragents that act by promoting glucose utilization, reducing hepaticglucose production and/or diminishing intestinal glucose output;alpha-glucosidase inhibitors such as acarbose and migiitoi) and otheragents that slow down carbohydrate digestion and consequently absorptionfrom the gut and reduce postprandial hyperglycemia; GLP-1 (glucagon likepeptide-1), GLP-1 analogs such as Exendin-4 and GLP-1 mimetics; andDPPIV (dipeptidyl peptidase IV) inhibitors such as vildagliptin;2. Hypolipidemic agents such as 3-hydroxy-3-methyl-glutaryl coenzyme A(HMG-CoA) reductase inhibitors, e.g. lovastatin, pitavastatin,simvastatin, pravastatin, cerivastatin, mevastatin, velostatin,fluvastatin, dalvastatin, atorvastatin, rosuvastatin and rivastatin;squalene synthase inhibitors; FXR (farnesoid X receptor) and LXR (liverX receptor) ligands; bile acid sequenstrants, such as cholestyramine andcolesevelam; fibrates; nicotinic acid and aspirin;3. Anti-obesity agents such as orlistat or rimonabant, phentermine,topiramate, qunexa, and locaserin;4. Anti-hypertensive agents, e.g. loop diuretics such as ethacrynicacid, furosemide and torsemide; angiotensin converting enzyme (ACE)inhibitors such as benazepril, captopril, enalapril, fosinopril,lisinopril, moexipril, perinodopril, quinapril, ramipril andtrandolapril; inhibitors of the Na—K-ATPase membrane pump such asdigoxin; neutralendopeptidase (NEP) inhibitors; ACE/NEP inhibitors suchas omapatrilat, sampatrilat and fasidotril; angiotensin II antagonistssuch as candesartan, eprosartan, irbesartan, losartan, telmisartan andvalsartan, in particular valsartan; renin inhibitors such as ditekiren,zankiren, terlakiren, aliskiren, RO 66-1132 and RO-66-1168; β-adrenergicreceptor blockers such as acebutolol, atenolol, betaxolol, bisoprolol,metoprolol, nadolol, propranolol, sotalol and timolol; inotropic agentssuch as digoxin, dobutamine and milrinone; calcium channel blockers suchas amlodipine, bepridil, diltiazem, felodipine, nicardipine, nimodipine,nifedipine, nisoldipine and verapamil; aldosterone receptor antagonists;and aldosterone synthase inhibitors;5. Agonists of peroxisome proliferator-activator receptors, such asfenofibrate, pioglitazone, rosiglitazone, tesaglitazar, BMS-298585,L-796449, the compounds specifically described in the patent applicationWO 2004/103995 i.e. compounds of examples 1 to 35 or compoundsspecifically listed in claim 21, or the compounds specifically describedin the patent application WO 03/043985 i.e. compounds of examples 1 to 7or compounds specifically listed in claim 19 and especially(R)-1-{4-[5-methyl-2-(4-trifluoromethyl-phenyl)-oxazol-4-ylmethoxy]-benzenesulfonyl}-2,3-dihydro-1H-indole-2-carboxylicor a salt thereof; and6. The specific anti-diabetic compounds described in Expert OpinInvestig Drugs 2003, 12(4): 623-633, FIGS. 1 to 7.

Furthermore, the present disclosure contemplates combination therapywith agents and methods for promoting weight loss, such as agents thatstimulate metabolism or decrease appetite, and modified diets and/orexercise regimens to promote weight loss.

EXAMPLE OF THE INVENTION Abbreviation

-   ACN Acetonitrile-   BEH Ethylene Bridged Hybrid-   BOC tert-Butyloxycarbonyl-   BSA Bovine serum albumin-   DCM dicloromethane-   DCC N,N′-dicyclohexylcarbodiimide-   DIC N,N′-Diisopropylcarbodiimide-   DIPEA N,N′-Diisopropylethylamine-   DMAP Dimethylaminopyridine-   DMF N,N′-Dimethylformamide-   DTT Dithiothreitol-   DOT 3,6-Dioxa-1,8-octanedithiol-   EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide-   EDTA ethylenediaminetretraacetic acid-   ESI electrospray ionization-   FFA fluorescent focus assay-   Fmoc fluorenylmethyloxycarbonyl chloride-   HCTU: O-(6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate-   HEP Heptane-   HFIP Hexafluoroisopropanol-   HPLC High performance liquid chromatography-   HRMS High resolution mass spectrometry-   HOBT Hydroxybenzotriazole-   HS Human serum-   LC/MS liquid chromatography/mass spectrometry-   MS Mass spectrometry-   MW molecular weight-   MRT mean residence time-   NHS N-hydroxysuccinimide-   NMM N-methylmorpholine-   NMR Nuclear magnetic resonance-   PEG polyethylene glycol-   pE Pyroglutamate-   pbf 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl-   PG: protective group-   PK pharmacokinetic-   Pol Polymer support-   QTOF: Quadrupole time-of-flight mass spectrometer-   Rt: retention time-   Rt or RT: room temperature-   Rpm: round per minute-   Sc subcutaneous-   SFC super critical fluid-   SPPS Solid phase peptide synthesis-   TBME methyl tert-butyl ether-   Trt trityl-   THF Tetrahydrofuran-   TEA trimethylamine-   TIS triethylsilane-   t, s, quin, br, m, d (triplet, singlet, quintet, broad, multiplet)-   UPLC Ultra performance liquid chromatography    Syntheses:    LCMS Methods Described

Method A Column Acquity BEH 1.7 μm 2.1 × 50 mm Column Temperature 50 C.Eluents A: Water (0.1% formic acid); B: ACN (0.1% formic acid) Flow Rate1 mL/min Gradient 0 min 2% B; 2% to 98% B in 1.7 min; 2.06 min 98% B;2.16 min 2% B Mass Spectrometer Single Quadrupole ESI scan range120-1600 UPLC Waters Acquity Method B Column Acquity BEH 1.7 μm 2.1 × 50mm Column Temperature 50 C. Eluents A: Water (0.1% formic acid); B: ACN(0.1% formic acid) Flow Rate 1 mL/min Gradient 0 min 40% B; 40% to 98% Bin 1.40 min; 2.05 min 98% B; 2.1 min 40% B Mass Spectrometer SingleQuadrupole ESI scan range 120-1600 UPLC Waters Acquity Method C ColumnXBridge C18 Column, 3.5 μm, 3.0 × 30 mm Column Temperature 40 C. EluentsA: Water (0.1% formic acid); B: ACN Flow Rate 2 mL/min Gradient 0 min40% B; 40% to 95% B in 1.70 min; 2.0 min 95% B; 2.1 min 40% B MassSpectrometer Single Quadrupole ESI scan range 150-1600 HPLC Agilent 1100series Method D Column Hilic 2.1 × 100 mm Column Temperature 55 C.Eluents A: CO₂ B: MeOH Flow Rate 2 mL/min Gradient 0.15 min 2% B; 2% to50% B in 1.5 min; 2.1 min 50% B; 2.25 min 2% B; 2.5 min 2% B MassSpectrometer Single Quadrupole ESI SCF Waters Acquity Method E ColumnProswift Monolith 4.6 × 50 mm Column Temperature 50 C. Eluents A: Water(0.1% formic acid); B: ACN (0.1% formic acid) Flow Rate 1 mL/minGradient 0.7 min 2% B; 2% to 60% B in 12.8 min; 14 min 60% B; 14.2 min2% B Mass Spectrometer Qtof ESI scan range 600-3500; deconvoluted by MaxEnt 1 in Mass Lynx software package UPLC Waters AcquityHPLC—Analytical Method F

-   -   Column: XBridge BEH300 C18 (100×4.6 mm), 3 μm; Part n°:        186003612    -   Eluent A: 0.1% TFA in water/Eluent B: 0.1% TFA in ACN    -   Flow: 1.0 ml/min    -   Temperature: 40° C.    -   Gradient:

Time [min] A [%] B [%] 0.0 98 2 18 2 98 20 2 98 22 98 2UPLC-HRMS—Analytic Method G

-   -   Waters Acquity UPLC® BEH C18, 1.7 μm, 2.1×50 mm; Part n°:        186002350    -   Eluent A: 0.05% FA +3.75 mM ammonium acetate in water; Eluent B:        0.04% FA in ACN    -   Flow: 1.0 ml/min    -   Temperature: 50° C.    -   Gradient: 2 to 98% in 4.4 min        Method H: LC-MS Method    -   HPLC: Mobile phase A: 2% HFIP+0.1% TEA; Mobile phase B:        Methanol;    -   Gradient: 0 min 95% A, 4 min: 75% A, 8 min 10% A, 8.1 min 95% A,        10 min 95% A;    -   Flow rate: 250 l/min;    -   Column: Acquity UPLC BEH C18, 1.7 um, 2.1×50 mm (waters);    -   Column temp: 75° C.    -   MS: QTOF (waters) negative mode;    -   ESI: 2.9 kv; Capillary temp 350° C.; Spray gas: 600 ml/min;        Source temp: 150° C.        Method I: LC-MS Method:    -   Mobile phase A: WATER+0.1% FORMIC ACID;    -   Mobile phase B: ACETONITRILE+0.1% FORMIC ACID;    -   Gradient: 0 min. 98% A, 0.06 min. 98% A, 1.76 min. 2% A, 2.06        min. 2% A, 2.16 min. 98%; Flow rate: 1 ml/min.;    -   Column: ACQUITY UPLC BEH C18, 130 Å, 1.7 μm, 2.1 mm×50 mm;    -   Column Temperature: 50° C.;    -   Detector: UV/Vis/CAD (charged Aerosol Detector)        UPLC HRMS Method J:    -   Column: Acquity BEH300 C4 1.7 μm, 2.1×50 mm    -   Eluent A: Water (0.1% TFA)    -   Eluent B: ACN (0.1% TFA)    -   Flow: 0.5 mL/min    -   Temperature: 40° C.    -   Gradient: 20% hold 0.5 min, ramp to 80% ACN in 10 min        Method K:    -   Column: Waters Protein BEH C4 Column, 300 Angstrom, 3.5 um,        4.6×100 mm    -   Mobile phase: A: Water (0.05% TFA) B: ACN (0.05% TFA)    -   Flow: 2 mL/min    -   Temperature: 40° C.    -   Gradient: Hold 25% B for 1 min, ramp from 25-60% ACN at 10 min,        ramp to 95% B at 10.50 min and hold for 2 mins, then equilibrate        at 25% for 2 min. Total runtime is 15 mins.    -   Mass Spectrometer: Waters ZQ mass spec    -   UPLC: Column: BEH C4, 300 Angstrom, 1.7 um, 2.1×50 mm        Method L:    -   Column: Proswift Monolith 4.6×50 mm    -   Mobile phase: A: Water (0.1% formic acid) B: ACN (0.1% formic        acid)    -   Flow: 1 mL/min    -   Temperature: 50° C.    -   Gradient: 0 min 3% B; 3% to 80% B in 2 min; 2.1 min 10% B; 2.8        min 95% B; 2.9 min 3% B    -   Mass Spectrometer: Qtof ESI scan range 100-1900; deconvoluted by        Max Ent 1 in Mass Lynx software package    -   UPLC: Waters Acquity

Method M Column Acquity BEH 1.7 μm 2.1 × 50 mm Column Temperature 50 C.Eluents A: Water (0.1% formic acid); B: ACN (0.1% formic acid) Flow Rate1 mL/min Gradient 0 min 2% B; 2% to 98% B in 4.40 min; 5.15 min 98% B;5.19 min 2% B Mass Spectrometer Single Quadrupole ESI scan range120-1600 UPLC Waters Acquity Method N Column Sunfire 30 × 50 mm 5 umEluents A: Water (0.1% TFA); B: ACN (0.1% TFA) Flow Rate 75 mL/minGradient 5-20% ACN over 3.2 min Method O Column Acquity BEH 1.7 μm 2.1 ×50 mm Column Temperature 50 C. Eluents A: Water (0.1% formic acid); B:ACN (0.1% formic acid) Flow Rate 1 mL/min Gradient 0 min 40% B; 40% to98% B in 3.40 min; 5.15 min 98% B; 5.19 min 40% B Mass SpectrometerSingle Quadrupole ESI scan range 120-1600 UPLC Waters Acquity Method PColumn Proswift Monolith 4.6 × 50 mm Column Temperature 50 C. Eluents A:Water (0.1% formic acid) B: ACN (0.1% formic acid) Flow Rate 1 mL/minGradient 0 min 2% B; 2% to 98% B in 2 min; 2.1 min 98% B; 2.3 min 2% B;3.3 min 2% B Mass Spectrometer Qtof ESI scan range 100-1900;deconvoluted by Max Ent 1 in Mass Lynx software package UPLC WatersAcquity Method Q Column Proswift Monolith 4.6 × 50 mm Column Temperature50 C. Eluents A: Water (0.1% formic acid); B: ACN (0.1% formic acid)Flow Rate 1 mL/min Gradient 0.7 min 2% B; 2% to 60% B in 12.8 min; 14min 60% B; 14.2 min 2% B Mass Spectrometer Qtof ESI scan range 600-3500;deconvoluted by Max Ent 1 in Mass Lynx software package UPLC WatersAcquity Method R Column Proswift Monolith 4.6 × 50 mm Column Temperature50 C. Eluents A: Water (0.1% formic acid) B: ACN (0.1% formic acid) FlowRate 1 mL/min Gradient 0 min 3% B; 3% to 90% B in 7 min; 7.1 min 15% B;7.70 min 95% B; 7.8 min 3% B Mass Spectrometer Qtof ESI scan range100-1900; deconvoluted by Max Ent 1 in Mass Lynx software package UPLCWaters AcquityAnalytical Method S:

-   -   Xbridge C18 Column, 3.5 μM, 3.0×3.0 mm    -   Eluent: A: Water+5 mM Ammonium Hydroxide B: ACN    -   Flow rate: 2 mL/min    -   Gradient: 0.0 min 2% B; 2% to 95% B in 1.70 min; 2.00 min 95% B;        2.10 min 5% B;    -   Mass Spectrometer: Single Quadrupole ESI    -   HPLC: Agilent 1100 series    -   Temperature: 40 C        Analytical Method T:

Column Acquity BEH 1.7 μm 2.1 × 50 mm Column 50 C. Temperature EluentsA: Water (0.1% formic acid) B: ACN (0.1% formic acid) Flow Rate 1 mL/minGradient 0 min 5% B; 5% to 60% B in 4 min; 7.2 min 98% B; 4.5 min 95% B;4.6 min 5% B Mass Acquity G2 Xevo QTof-Rs (FWHM) >20000 AccuracySpectrometer <5 ppm UPLC Waters Acquity

Intermediate 1: Benzyl 11-bromoundecanoate

11-bromoundecanoic acid (4 g, 15.08 mmol), benzyl alcohol (1.875 mL,18.10 mmol), and DMAP (92 mg, 0.754 mmol) were dissolved in DCM under N₂at room temperature. EDC-HCl (4.34 g, 22.63 mmol) was added and thereaction stirred for 17 hr. The reaction was concentrated, followed bydilution with Et₂O (150 mL). The mixture was extracted with water (30mL), and the aqueous phase extracted with Et₂O (150 mL). The combinedorganics were washed with brine (20 mL) and dried over Na2SO4. Thesolvent was removed and the residue purified by flash column (silica 120g, 0-10% Et₂O/petroleum ether) to yield intermediate 1 as a colorlessliquid (6.75 g, quantitative): LCMS method Method A Rt=1.79 min, M+H355.2; ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.18-1.36 (m, 10H) 1.37-1.47(m, 2H) 1.64 (quin, J=7.33 Hz, 2H) 1.85 (dt, J=14.56, 7.06 Hz, 2H) 2.35(t, J=7.58 Hz, 2H) 3.40 (t, J=6.88 Hz, 2H) 5.11 (s, 2H) 7.28-7.45 (m,5H).

Intermediate 2: Tribenzyl Undecane-1,1,11-tricarboxylate

NaH (113 mg, 2.83 mmol) was suspended in DMF (6 mL) under N2 at 0° C.Dibenzyl malonate (0.704 mL, 2.82 mmol) was slowly added to the stirringsuspension over 30 min. intermediate 1 (903 mg, 2.54 mmol) dissolved inDMF (3 mL) was added and the reaction allowed to stir at 0° C. for 2.75hr before being allowed to warm to room temperature and stir overnight.The reaction was diluted with Et₂O (75 mL) and extracted with water (20mL). The aqueous phase was extracted with Et₂O (75 mL) and the combinedorganics washed with brine (30 mL). The organics were dried over Na2SO4and concentrated. The concentrate was purified by flash column (silica80 g, 0-10% EtOAc/HEP) to yield a colorless oil (770 mg, 1.38 mmol, 34%)of 70% purity: LCMS Method B Rt=1.41 min, M+H 559.6.

Intermediate 3: Tribenzyl Docosane-1,11,11-tricarboxylate

To a suspension of NaH (66.1 mg, 1.65 mmol) in DMF (2 mL) at 0° C. underN2, was added Intermediate 2 (770 mg, 1.38 mmol) in DMF (4 mL). After 35min a solution of 1-bromoundecane (0.338 mL, 1.52 mmol) in DMF (2 mL)was added to the reaction, which was allowed to warm to room temperatureafter stirring for 25 min. The reaction was stirred for 2 days. Thereaction was diluted with Et₂O (75 mL) and extracted with 10% LiCl (25mL). The aqueous phase was extracted with Et₂O (75 mL). The combinedorganics were washed with brine, dried over Na2SO4, and the solventevaporated. Purification of the residue by flash column (silica 80 g,0-10% EtOAc/HEP) yielded intermediate 3 as a colorless oil (590 mg,0.827 mmol, 33%): LCMS method Method B Rt=1.89 min, M+Na 735.5; ¹H NMR(400 MHz, CHLOROFORM-d) δ ppm 0.87-0.95 (m, 3H) 1.07 (br. s., 4H)1.14-1.36 (m, 28H) 1.66 (quin, J=7.43 Hz, 2H) 1.85-1.95 (m, 4H) 2.37 (t,J=7.58 Hz, 2H) 5.12 (s, 4H) 5.14 (s, 2H) 7.27 (d, J=2.32 Hz, 1H)7.28-7.43 (m, 14H).

Intermediate 4: Docosane-1,11,11-tricarboxylic Acid

Intermediate 3 (590 mg, 0.827 mmol) dissolved in THF (12 mL) wascombined with a suspension of 10% Pd on carbon in THF (8 mL). Thesuspension was stirred and placed under a hydrogen atmosphere viaballoon. After 1 hr the reaction was passed through a membrane filterand the solids rinsed with EtOAc. The filtrate was evaporated, yieldingthe title compound as a colorless oil (353 mg, 0.798 mmol, 96%): LCMSmethod Method B Rt=1.16 min, M+H 443.5; ¹H NMR (400 MHz, CHLOROFORM-d) δppm 0.77-0.84 (m, 3H) 1.06-1.33 (m, 32H) 1.59 (quin, J=7.18 Hz, 2H)1.83-1.92 (m, 4H) 2.32 (t, J=7.03 Hz, 2H).

Intermediate 5:2-(((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)-2-undecyltridecanedioic Acid

A solution of DCC (126 mg, 0.610 mmol) in DCM (1.57 mL) was added to asolution of intermediate 4 and N-hydroxysuccinimide in DCM (5 mL) andTHF (5 mL) under N₂. After 3.5 hrs the solvent was evaporated and theresidue purified by supercritical fluid chromatography (SFC; Princeton2-ethyl-pyridine, 20×150 mm, 20-30% MeOH/CO₂), yielding the titlecompound as a colorless oil (138 mg, 0.256 mmol, 50%): LCMS method BRt=1.21 min, M+H 540.5; ¹H NMR (600 MHz, ACETONITRILE-d₃) δ ppm 0.91 (t,J=7.20 Hz, 3H) 1.22-1.42 (m, 34H) 1.57 (quin, J=7.34 Hz, 2H) 1.93-1.96(m, 2H) 2.28 (t, J=7.47 Hz, 2H) 2.79 (br. d, J=6.30 Hz, 4H).

Intermediate 6 and 6A: 2-(Azido-PEG23-carbamoyl)-2-undecyltridecanedioicAcid Construct (6) and 12-(Azido-PEG23-cabamoyl)tricosanoic AcidConstruct (6A)

Intermediate 5 (36 mg, 0.066 mmol) and azido-dPEG23—NH₂ (QuantaBiodesign: 73 mg, 0.066 mmol) were combined in THF (1.5 mL) and mixed ona shaker plate for 15 min before addition of DIPEA (17 μL, 0.10 mmol).The reaction was left on the shaker plate overnight. The solvent wasevaporated and the residue purified by HPLC (Sunfire C18 30×50 mm,55-80% ACN/water+0.1% TFA) to yield Intermediate 6 (39 mg, 0.025 mmol,38%) and intermediate 6a (20 mg, 0.013 mmol, 20%): LCMS Method B Rt=1.11min, [M+2H]⁺² 763.4; ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.86-0.93 (m,3H) 1.10-1.19 (m, 2H) 1.20-1.29 (m, 23H) 1.32 (br. s., 7H) 1.58-1.69 (m,2H) 1.69-1.79 (m, 2H) 1.96-2.10 (m, 2H) 2.35 (t, J=7.15 Hz, 2H) 3.41 (t,J=5.07 Hz, 2H) 3.51-3.57 (m, 2H) 3.58-3.62 (m, 2H) 3.62-3.73 (m, 90H)7.46 (br. s., 1H); LCMS Method B Rt=1.23 min, [M+2]⁺² 740.9; ¹H NMR (400MHz, CHLOROFORM-d) δ ppm 0.83-0.96 (m, 3H) 1.27 (br. s., 25H) 1.29-1.37(m, 7H) 1.37-1.46 (m, 2H) 1.53-1.73 (m, 4H) 2.34 (t, J=7.21 Hz, 2H) 3.41(t, J=5.07 Hz, 2H) 3.44-3.52 (m, 2H) 3.55-3.60 (m, 2H) 3.60-3.74 (m,90H) 6.19-6.30 (m, 1H).

Alternatively construct 6 A is obtained according to the followingprocedure: A solution of intermediate 5 (48 mg, 0.042 mmol) in THF (1mL) was added to a vial charged with azido-PEG23-amine (Quanta Biodesigncat #10525) (46 mg, 0.042 mmol). The reaction was agitated for 20 minbefore the addition of DIPEA (11 μL, 0.063 mmol) and then maintainedovernight. Azido-PEG23-amine (23 mg, 0.021 mmol) and DIPEA (5 μL, 0.029mmol) were added and the reaction agitated another day. The solvent wasevaporated and the residue purified by HPLC (Xbridge C18 30×50 mm,10-30% ACN/5 mM NH₄OH). Lyophilization of the fractions resulted in amixture of products. The material was purified by HPLC (Sunfire C1830×50 mm, 45-70% ACN/water+0.1% TFA) to yield the title intermediate 6A(30 mg, 0.020 mmol, 48%): LCMS method B Rt=0.81 min, [M+H+H₃O]⁺² 764.5;¹H NMR (400 MHz, ACETONITRILE-d₃) δ ppm 1.30 (br. s., 28H) 1.40-1.50 (m,2H) 1.50-1.62 (m, 6H) 2.14 (t, J=7.52 Hz, 2H) 2.23-2.35 (m, 3H) 3.32 (q,J=5.58 Hz, 2H) 3.37-3.43 (m, 2H) 3.47-3.52 (m, 2H) 3.53-3.68 (m, 90H)6.54 (br. s., 1H).

Intermediate 7: (((11-Bromoundecyl)oxy)methanetriyl)tribenzene

Trityl chloride (2.49 g, 8.92 mmol), 11-bromoundecan-1-ol (2.00 g, 7.96mmol), and DMAP (10 mg, 0.080 mmol) were dissolved in DCM (16 mL) underN₂. With stirring DIPEA (1.39 mL, 7.96 mmol) was added and the reactionwas maintained for 7 days. The reaction was partitioned between DCM (20mL) and water (10 mL). The organic phase was extracted with water (20mL), dried over MgSO₄, and concentrated. The concentrate was purified byflash column (silica 120 g, 0-6% EtOAc/HEP) to yield Intermediate 7(2.50 g, 5.07 mmol, 64%) as a colorless oil: 1H NMR (400 MHz,CHLOROFORM-d) δ ppm 1.19-1.49 (m, 14H) 1.58-1.69 (m, 2H) 1.79 (dt,J=14.50, 7.00 Hz, 1H) 1.87 (dt, J=14.55, 7.03 Hz, 1H) 3.07 (t, J=6.66Hz, 2H) 3.43 (t, J=6.85 Hz, 1H) 3.55 (t, J=6.79 Hz, 1H) 7.18-7.36 (m,10H) 7.42-7.52 (m, 5H).

Intermediate 8: Dibenzyl 2-(11-(trityloxy)undecyl)malonate

NaH (113 mg, 2.83 mmol) was suspended in DMF (6 mL) at 00° C. under N2.Dibenzyl malonate was slowly added to the stirred suspension. After 30min a solution of Intermediate 7 (1.26 g, 2.54 mmol) in DMF (3 mL) wasadded. After 15 min of stirring, the resulting mixture was allowed towarm to room temperature. After 3 days the reaction was diluted withEt₂O (75 mL) and extracted with water (40 mL). The aqueous phase wasextracted with Et₂O (75 mL). The combined organics were dried overNa₂SO₄ and concentrated. The concentrate was purified by flash column(silica 80 g, 0-10% EtOAc/HEP) to yield the title compound as acolorless oil (815 mg, 1.17 mmol, 41%): HPLC Method B Rt=1.68 min; ¹HNMR (400 MHz, CHLOROFORM-d) δ ppm 1.16-1.40 (m, 16H) 1.58-1.69 (m, 2H)1.94 (q, J=7.38 Hz, 2H) 3.06 (t, J=6.66 Hz, 2H) 3.45 (t, J=7.52 Hz, 1H)5.16 (s, 4H) 7.21-7.28 (m, 3H) 7.28-7.39 (m, 16H) 7.42-7.51 (m, 6H).

Intermediate 9: Tribenzyl 22-(trityloxy)docosane-1,11,11-tricarboxylate

A solution of intermediate 8 (815 mg, 1.17 mmol) in DMF (2 mL) was addedto a suspension of NaH (56 mg, 1.40 mmol) in DMF (2 mL) under N₂ at 0°C. The mixture was stirred for 1 hr. Benzyl 11-bromoundecanoate (457 mg,1.29 mmol) in DMF (2 mL) was added to the reaction. The reaction wasallowed to warm to room temperature 20 min after the addition andstirred overnight. The reaction was diluted with Et₂O (75 mL) andextracted with water (25 mL). The aqueous phase was extracted with Et₂O(75 mL) and the organics combined. The combined organics were dried overNa₂SO₄ and evaporated. The residue was purified by flash column (silica40 g, 0-10% EtOAc/HEP) to yield the title compound as a colorless oil(780 mg, 0.803 mmol, 69%): ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm0.99-1.14 (m, 4H) 1.15-1.41 (m, 26H) 1.58-1.71 (m, 4H) 1.82-1.96 (m, 4H)2.37 (t, J=7.52 Hz, 2H) 3.06 (t, J=6.66 Hz, 2H) 5.12 (s, 4H) 5.14 (s,2H) 7.28 (s, 24H) 7.42-7.52 (m, 6H).

Intermediate 10: 22-(Ttrityloxy)docosane-1,11,11-tricarboxylic Acid

A suspension of 10% Pd on carbon (11 mg, 0.010 mmol) in THF (2.5 mL) wasadded to a solution of intermediate 9 (200 mg, 0.206 mmol) in THF (2.5mL). The stirred suspension was placed under hydrogen via balloon. After2.25 hrs the reaction was passed through a membrane filter and thesolids rinsed with EtOAc. The filtrate was evaporated to yieldintermediate 10 (150 mg, quantitative): ¹H NMR (400 MHz, CHLOROFORM-d) δppm 1.04-1.33 (m, 30H) 1.45-1.62 (m, 4H) 1.76-1.91 (m, 4H) 2.21-2.36 (m,2H) 2.97 (t, J=6.60 Hz, 2H) 7.06-7.18 (m, 4H) 7.19-7.24 (m, 5H)7.33-7.50 (m, 6H).

Intermediate 11:2-(((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)-2-(11-(trityloxy)undecyl)tridecanedioicAcid (11)

Intermediate 10 (150 mg, 0.214 mmol) and N-hydroxysuccinimide (25 mg,0.214 mmol) were combined in DCM (4 mL). DCC (49 mg, 0.235 mmol)dissolved in DCM (0.61 mL) was added, and the reaction stirred at roomtemperature for 7 hrs. The solvent was evaporated and the residuepurified by HPLC (Sunfire C18 30×50 mm; 65-95% ACN/water+0.1% TFA)followed by SFC (Princeton 2-ethylpyridine column 20×100 mm, 25-35%MeOH/CO2) to yield Intermediate 11 (34 mg, 0.043 mmol, 20%) as acolorless oil: LCMS Method B Rt=1.47 min, M-CO₂H 752.7; ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 1.13-1.42 (m, 30H) 1.56-1.73 (m, 4H) 1.94-2.14 (m,4H) 2.37 (t, J=7.21 Hz, 2H) 2.83 (br. s., 4H) 3.06 (t, J=6.66 Hz, 2H)7.15-7.28 (m, 3H) 7.29-7.36 (m, 6H) 7.41-7.50 (m, 6H).

Intermediate 12:2-((Azido-PEG23)carbamoyl)-2-(11-hydroxyundecyl)tridecanedioic AcidConstruct

Azido-dPEG23-amine (Quanta Biodesign) 42 mg, 0.038 mmol) in THF (1.5 mL)was combined with intermediate 11 (34 mg, 0.043 mmol) under N₂. Thereaction was placed on a shaker plate and agitated for 20 min. DIPEA(7.44 μL, 0.043 mmol) was added and the reaction agitated for 2 hrs.DIPEA (4 μL, 0.023 mmol) was added and the reaction maintainedovernight. The solvent was evaporated and the residue taken up in DCM (3mL) and TFA (0.5 mL). The solution was agitated for 1 hr at which pointthe solvent was stripped. The residue was purified by HPLC (sunfire C1830×50 mm, 45-70% ACN/water+0.1% TFA) to yield intermediate 12 (4 mg, 1.8μmol, 4.2%): LCMS Method B Rt=0.75 min, [M+2H]⁺² 771.4; ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 1.05-1.39 (m, 30H) 1.52-1.82 (m, 6H) 1.97-2.09 (m,2H) 2.35 (t, J=7.21 Hz, 2H) 3.41 (t, J=5.07 Hz, 2H) 3.51-3.63 (m, 6H)3.63-3.75 (m, 90H) 4.36 (t, J=6.72 Hz, 1H) 7.49-7.65 (m, 1H).

Intermediate 13:13-(Benzyloxy)-12-((benzyloxy)carbonyl)-13-oxotridecanoic Acid

Dibenzyl malonate (0.88 mL, 3.52 mmol) in DMF (3 mL) was slowly added toa suspension of NaH (274 mg, 6.86 mmol) under N₂ at 00° C. The mixturewas stirred for 1.5 hrs before being allowed to warm to room temperate.11-bromoundecanoic acid (933 mg, 3.52 mmol) in DMF (3 mL) was added andthe reaction allowed to go overnight. The reaction was heated to 80° C.for 3 hrs before being allowed to cool. The reaction was diluted withEtOAc (50 mL) and Et₂O (50 mL) and extracted with 1M HCl (25 mL). Theaqueous phase was extracted with EtOAc/Et₂O (100 mL). The combinedorganics were dried over Na₂SO₄ and the solvent evaporated. The residuewas purified by flash column (C18 50 g 30-100% ACN/water+0.1 TFA) toyield intermediate 13 (315 mg, 0.672 mmol, 19%) as white powder: LCMSMethod B Rt=1.05 min, M+H 469.5.

Intermediate 14:23-(Benzyloxy)-12,12-bis((benzyloxy)carbonyl)-23-oxotricosanoic Acid

NaH (54 mg, 1.34 mmol) was suspended in DMF (1 mL) at 00° C. under N₂.To the mixture was added intermediate 13 (315 mg, 0.672 mmol) in DMF (3mL) in a drop wise fashion. The reaction was stirred for 1 hr beforeintermediate 1 (239 mg, 0.672 mmol) in DMF (1 mL) was added. Thereaction was maintained at 00° C. for an additional 45 min before beingallowed to warm to room temperature. The reaction was stirred aovernight. The reaction was diluted with 1:1 Et₂O and EtOAc (75 mL) andextracted with 1M HCl (20 mL). The aqueous phase was extracted with 1:1Et₂O and EtOAc (75 mL). The combined organics were dried over Na₂SO₄ andevaporated. Purification of the resulting residue by HPLC (Xbridge C1830×50 mm, 45-70% ACN/water+5 mM NH₄OH) yielded the title compound (132mg, 0.178 mmol, 26%): LCMS method B; Rt=1.53 min, M−H 741.8.

Intermediate 15: 1,11,11-Tribenzyl 21-(2,5-dioxopyrrolidin-1-yl)Henicosane-1,11,11,21-tetracarboxylate

DCC (44 mg, 0.213 mmol) in DCM (1 mL) was added to a solution ofintermediate 14 (132 mg, 0.178 mmol) and of N-hydroxysuccinimide (20 mg,0.178 mmol) in DCM (2.5 mL). The reaction was agitated on a shaker platefor 17 hrs. The reaction was filtered and the solids rinsed with DCM.The filtrate was concentrated and purified by flash column (silica 12 g,0-40% EtOAc/HEP) to yield intermediate 15 (107 mg, 0.127 mmol, 72%):LCMS Method B Rt=1.53 min, M+Na 862.8.

Intermediate 16:22-((2,5-Dioxopyrrolidin-1-yl)oxy)-22-oxodocosane-1,11,11-tricarboxylicAcid

To a solution of intermediate 15 (107 mg, 0.127 mmol) in THF (2.5 mL)was added a suspension of 10% Pd on carbon in THF (2.5 mL). The mixturewas placed under a hydrogen atmosphere for 1.5 hrs. The reaction waspassed through a membrane filter and the solids washed with DCM and THF.The filtrate was evaporated to yield a colorless oil (95 mg,quantitative) which contained the title compound: LCMS Method B Rt=0.65min, M+H 570.5.

Intermediate 17: 2-(11-(azido-PEG23-amino)-11-oxoundecyl)tridecanedioicAcid Construct

A solution of intermediate 16 (48 mg, 0.042 mmol) in THF (1 mL) wasadded to a vial charged with azido-dPEG23-amine (Quanta Biodesign: 46mg, 0.042 mmol). The reaction was agitated for 20 min before theaddition of DIPEA (11 μL, 0.063 mmol) and then maintained overnight.Azido-PEG23-amine (23 mg, 0.021 mmol) and DIPEA (5 μL, 0.029 mmol) wereadded and the reaction agitated another day. The solvent was evaporatedand the residue purified by HPLC (Xbridge C18 30×50 mm, 10-30% ACN/5 mMNH₄OH). Lyophilization of the fractions resulted in a mixture ofproducts. The material was purified by HPLC (Sunfire C18 30×50 mm,45-70% ACN/water+0.1% TFA) to yield the title intermediate 17 (30 mg,0.020 mmol, 48%): LCMS SQ4 Rt=0.81 min, [M+H+H₃O]⁺² 764.5; ¹H NMR (400MHz, ACETONITRILE-d₃) δ ppm 1.30 (br. s., 28H) 1.40-1.50 (m, 2H)1.50-1.62 (m, 6H) 2.14 (t, J=7.52 Hz, 2H) 2.23-2.35 (m, 3H) 3.32 (q,J=5.58 Hz, 2H) 3.37-3.43 (m, 2H) 3.47-3.52 (m, 2H) 3.53-3.68 (m, 90H)6.54 (br. s., 1H).

Intermediate 18: Tetrabenzyl henicosane-1,11,11,21-tetracarboxylate

To a suspension of NaH (48 mg, 1.21 mmol) in DMF (2 mL) at 00° C. underN₂, was slowly added intermediate 2 (337 mg, 0.603 mmol) in DMF (2 mL).The mixture was stirred for 15 min before the addition of intermediate 1(429 mg, 1.21 mmol) in DMF (2 mL). The reaction was stirred at 00° C.for 20 min before being allowed to warm to room temperature. Thereaction was maintained at room temperature with stirring for 3 days.The reaction was diluted with Et₂O (75 mL) and extracted with water (20mL). The aqueous phase was extracted with Et₂O (75 mL). The combinedorganics were dried over Na₂SO₄ and evaporated. The residue was purifiedby flash column (silica 24 g, 0-15% EtOAc/HEP) to yield the titlecompound (315 mg, 0.378 mmol, 63%): LCMS Method B Rt=1.70 min, M+Na855.8; ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.95-1.13 (m, 4H) 1.13-1.40(m, 24H) 1.59-1.72 (m, 4H) 1.82-1.95 (m, 4H) 2.37 (t, J=7.52 Hz, 4H)5.12 (s, 4H) 5.14 (s, 4H) 7.14-7.44 (m, 20H).

Intermediate 19: Henicosane-1,11,11,21-tetracarboxylic Acid

A suspension of 10% Pd on carbon (20 mg, 0.019 mmol) in THF (4 mL) wasadded to a solution of intermediate 18 (315 mg, 0.378 mmol) in THF (6mL), and the reaction was placed under a hydrogen atmosphere for 2 hr. Amembrane filter was used to remove the solids which were washed withEtOAc. Evaporation of the filtrate yielded intermediate 19 (179 mg,quantitative) as a white solid: LCMS Method A Rt=1.24 min, M+H 473.4; ¹HNMR (400 MHz, DMSO-d₆) δ ppm 0.99-1.15 (m, 4H) 1.24 (br. s., 24H) 1.48(quin, J=6.94 Hz, 4H) 1.62-1.76 (m, 4H) 2.18 (t, J=7.34 Hz, 4H) 12.23(br. s, 4H).

Intermediate 20:11-(((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)henicosane-1,11,21-tricarboxylicAcid

N-hydroxysuccinimide (20 mg, 0.170 mmol) and intermediate 19 (90 mg,0.190 mmol) were dissolved in DCM (3 mL) and THF (0.3 mL). A solution ofDCC (39 mg, 0.190 mmol) in DCM (0.5 mL) was added and the reactionagitated overnight. The solvent was evaporated and the residue purifiedby HPLC (Sunfire C18 30×50 mm; 35-60% ACN/water+0.1% TFA) to yield thetitle compound as a white powder (21 mg, 0.037 mmol, 19%): LCMS (MethodC Rt=1.01 min, M+H 570.3.

Intermediate 21 and 21a: 11-((Azido-PEG23)carbamoyl)Henicosane-1,11,21-tricarboxylic Acid (21) and12-((Azido-PEG23)carbamoyl) tricosanedioic Acid (21a)

Azido-PEG23-amine (41 mg, 0.037 mmol) and intermediate 20 (21 mg, 0.037mmol) were combined in THF (1 mL) and agitated for 10 min. DIPEA (9.66μL, 0.055 mmol) was added, and the reaction was agitated overnight. Thesolvent was evaporated, and the residue purified by HPLC (Sunfire C1830×50 mm, 35-60% ACN/water+0.1% TFA) to yield intermediate 21 (22 mg,0.014 mmol, 38%) and 21a (4 mg, 2.6 mol, 7%): LCMS Method B Rt=0.69 min,M+H 1555.3; ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.27 (br. s., 19H)1.29-1.41 (m, 9H) 1.65 (quin, J=7.12 Hz, 4H) 1.78 (td, J=12.13, 4.22 Hz,2H) 1.95-2.08 (m, 2H) 2.35 (t, J=7.21 Hz, 4H) 3.41 (t, J=5.07 Hz, 2H)3.54 (q, J=5.05 Hz, 2H) 3.58-3.77 (m, 92H) 7.60 (t, J=4.95 Hz, 1H); LCMSMethod B Rt=0.78 min, M−H 1509.3; ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm1.18 (br. s., 19H) 1.21-1.38 (m, 11H) 1.43-1.63 (m, 6H) 1.91-2.04 (m,1H) 2.26 (t, J=7.15 Hz, 4H) 3.31 (t, J=5.07 Hz, 2H) 3.40 (q, J=5.14 Hz,2H) 3.46-3.50 (m, 2H) 3.51-3.69 (m, 90H) 6.23 (t, J=5.01 Hz, 1H).

Intermediate 22: Dibenzyl 2-undecylmalonate

Dibenzyl malonate (0.88 mL, 3.52 mmol) in DMF (1.5 mL) was added dropwise to a suspension of NaH (155 mg, 3.87 mmol) in DMF (6 mL) under N2at 00° C. The mixture was stirred for 30 min before the addition of1-bromoundecane (0.785 mL, 3.52 mmol) in DMF (1.5 mL) to the reaction.The reaction was allowed to warm to room temperature and stirred for 5days. The reaction was diluted with Et₂O (75 mL) and extracted withwater (20 mL). The aqueous phase was extracted with Et₂O (75 mL). Thecombined organics were dried over Na₂SO₄ and evaporated. The residue waspurified by flash column (silica 80 g, 0-10% EtOAc/HEP) to yield thetitle compound (974 mg, 2.22 mmol, 63%) as a colorless oil: LCMS MethodB Rt=1.55 min, M+H 439.5.

Intermediate 23:2-(((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)-2-undecyltridecanoic Acid

Dibenzyl 2-undecylmalonate (Intermediate 22: 400 mg, 0.912 mmol) in DMF(1 mL) was added drop wise to a suspension of NaH (44 mg, 1.09 mmol) inDMF (2 mL) under N2 at 00° C. The mixture was stirred for 45 min beforethe addition of 1-bromoundecane (0.285 mL, 1.28 mmol) in DMF (1 mL) tothe reaction. The reaction was allowed to warm to room temperature andstirred for 1 day. The reaction was diluted with Et₂O (75 mL) andextracted with water (20 mL). The aqueous phase was extracted with Et₂O(75 mL). The combined organics were dried over Na₂SO₄ and evaporated.The residue was purified by flash column (silica 40 g, 0-5% EtOAc/HEP)to yield a colorless oil (412 mg). The oil was dissolved in THF/MeOH andpassed through a Thales Nano H-Cube (1 mL/min, 2 bar H2, 22C) with a 10%Pd/C cartridge. The effluent was collected and evaporated to yield awaxy solid (272 mg). The waxy solid was dissolved in 3:1 DCM/THF andconcentrated to an oil. The oil was redissolved in DCM (6 mL) under N₂,and N-hydroxysuccinimide (68 mg), followed by DCC (136 mg) in DCM (3mL), was added. The reaction was stirred for day. The reaction wasfiltered and the filtrate concentrated. The concentrate was purified byflash column (C18 12 g, 25-100% ACN/water+0.1% formic acid). Theresulting material was purified further by supercritical fluidchromatography (Princeton 2-ethylpyridine 20×150 mm; 5-15% MeOH/CO₂) toyield intermediate 23 (37 mg, 0.073 mmol, 8%): LCMS Method B Rt=1.67min, M+NH₄ 527.6; ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.80-0.94 (m, 6H)1.18-1.42 (m, 36H) 1.97-2.14 (m, 4H) 2.87 (br. s., 4H).

Intermediate 24: 2-((azido-PEG23)carbamoyl)-2-undecyltridecanoic Acid

A solution of intermediate 23 (37 mg, 0.073 mmol) in THF (1 mL) wasadded to a vial charged with azido-dPEG23-amine (Quanta Biodesign: 80mg, 0.073 mmol). The solution was agitated on a shaker plate and DIPEA(11 L, 0.065 mmol) added. The reaction was agitated overnight before anadditional portion of DIPEA (12 μL, 0.071 mmol) was added, and thereaction allowed to go overnight. The solvent was evaporated and theresidue purified by supercritical fluid chromatography (Princeton Amino21×150 mm; 20-30% MeOH/CO₂) to yield the title compound (45 mg, 0.030mmol, 41%): LCMS Method B Rt=1.50 min, [M+2H]⁺² 748.1; ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 0.90 (t, J=6.85 Hz, 6H) 1.09-1.38 (m, 30H) 1.58 (br.s., 12H) 1.64-1.76 (m, 2H) 1.98-2.16 (m, 2H) 3.41 (t, J=5.14 Hz, 2H)3.46-3.64 (m, 5H) 3.64-3.91 (m, 83H).

Intermediate 25: Di-tert-butyl 2-undecylmalonate

Di-tert-butyl malonate (1.0 g, 4.62 mmol) in DMF (2 mL) was added to asuspension of NaH (213 mg, 5.32 mmol) in DMF (5 mL) under N₂ at 00° C.The reaction was stirred for 30 min before the addition of1-bromoundecane in DMF (2 mL). Upon addition the reaction was allowed towarm to room temperature and stirred for 2 days. The reaction wasdiluted with Et₂O (75 mL) and extracted with water (25 mL). The aqueousphase was extracted with Et₂O (75 mL). The combined organics were driedover Na₂SO₄ and the solvent evaporated. The concentrate was purified byflash column (silica 120 g, 0-40% Et₂O/petroleum ether) to yieldintermediate 25 (0.998 g, 2.69 mmol, 58%): LCMS Method B Rt=1.64 min,M+Na 393.5; ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.85-0.94 (m, 3H)1.24-1.36 (m, 18H) 1.41-1.52 (m, 18H) 1.74-1.86 (m, 2H) 3.13 (t, J=7.58Hz, 1H).

Intermediate 26: 1-Benzyl 11,11-di-tert-butylDocosane-1,11,11-tricarboxylate

The title compound was synthesized in a similar fashion as intermediate9 using intermediate 25 as a starting material to yield a colorless oil(980 mg, 1.52 mmol, 66%): ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.89 (t,J=6.91 Hz, 3H) 1.06-1.20 (m, 4H) 1.20-1.35 (m, 28H) 1.45 (s, 18H)1.58-1.70 (m, 2H) 1.72-1.83 (m, 4H) 2.36 (t, J=7.52 Hz, 2H) 5.12 (s, 2H)7.30-7.45 (m, 5H).

Intermediate 27: 12,12-Bis(tert-butoxycarbonyl)tricosanoic Acid

Using intermediate 26, the title compound (472 mg, 0.851 mmol, 100%) wassynthesized in a similar fashion as intermediate 19: LCMS Method BRt=1.76 min, M−H 553.6; ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.84-0.95(m, 3H) 1.07-1.21 (m, 4H) 1.21-1.40 (m, 28H) 1.46 (s, 18H) 1.58-1.70 (m,2H) 1.72-1.84 (m, 4H) 2.37 (t, J=7.46 Hz, 2H).

Intermediate 28: 11,11-Di-tert-butyl 1-(2,5-dioxopyrrolidin-1-yl)Docosane-1,11,11-tricarboxylate

Intermediate 27 (200 mg, 0.360 mmol) and N-hydroxysuccinimide (42 mg,0.360 mmol) were dissolved in DCM (3 mL). A solution of DCC (89 mg,0.433 mmol) in DCM (1.6 mL) was added and the reaction stirred for 4.5hrs. The reaction was filtered and the filtrate concentrated. Theconcentrate was purified by flash column (silica 24 g, 0-40% EtOAc/HEP)to yield the title compound as a colorless oil (70 mg, 0.107 mmol, 30%):LCMS Method B Rt=1.74 min, M+Na 674.7.

Intermediates 29 and 29A:2-(11-((azido-PEG23)-amino)-11-oxoundecyl)-2-undecylmalonic Acid (29)and 13-((azido-PEG23)-amino)-13-oxo-2-undecyltridecanoic Acid (29A)

A solution of Intermediate 28 (35 mg, 0.054 mmol) in THF (1 mL) wasadded to a vial charged with azido-PEG23-amine (59 mg, 0.054 mmol).DIPEA (14 L, 0.081 mmol) was added and the reaction was agitatedovernight. The solvent was evaporated and the residue redissolved in DCM(1 mL) and TFA (0.2 mL). The reaction was agitated for 1.25 hr beforethe solvent was evaporated. The residue was purified by HPLC (Sunfire30×50 mm C18, 55-80% ACN/water+0.1% TFA) and the resulting material wasredissolved in DCM (4 mL) and TFA (2 mL) and agitated for a 1.5 hrs. Thesolvent was evaporated and the residue purified by HPLC (Sunfire 30×50mm C18, 55-80% ACN/water+0.1% TFA) to yield intermediate 29 (28 mg,0.016 mmol, 29%) and intermediate 29A (1 mg, 0.6 μmol, 1%): LCMS MethodB Rt=1.08 min, [M+H+H₃O]⁺² 771.5; ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm0.90 (t, J=6.72 Hz, 3H) 1.26 (br. s., 24H) 1.32-1.41 (m, 8H) 1.62 (quin,J=7.64 Hz, 2H) 1.88-2.01 (m, 4H) 2.31 (t, J=7.70 Hz, 2H) 3.41 (t, J=5.07Hz, 2H) 3.46-3.56 (m, 3H) 3.57-3.90 (m, 91H); LCMS Method B Rt=1.29 min,[M+2H]⁺² 741.1.

Intermediate 30:22-((azido-PEG23)amino)-22-oxodocosane-1,11,11-tricarboxylic Acid

A solution of intermediate 16 (58 mg, 0.063 mmol) in THF (1 mL) wasadded to a vial charged with azido-PEG23-amine (70 mg, 0.063 mmol).DIPEA (17 μL, 0.095 mmol) was added and the reaction agitated on ashaker plate overnight. The reaction was concentrated and purified byHPLC (Sunfire C18 30×50 mm, 35-60% ACN/water+0.1% TFA) to yieldintermediate 30 (57 mg, 0.036 mmol, 57%) as waxy white solid: LCMSMethod B Rt=0.62 min, M+H 1555.4; ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm1.28 (br. s., 18H) 1.30-1.40 (m, 10H) 1.63 (m, J=7.10, 7.10, 7.10, 7.10,7.10 Hz, 4H) 1.88-2.02 (m, 4H) 2.28 (t, J=8.10 Hz, 2H) 2.35 (t, J=7.40Hz, 2H) 3.41 (t, J=5.07 Hz, 2H) 3.50 (dt, J=9.20, 4.39 Hz, 2H) 3.57-3.63(m, 2H) 3.63-3.73 (m, 90H)

Intermediate 31: 1-Benzyl 3-tert-butyl 2-undecylmalonate

To a suspension of NaH (160 mg, 4.0 mmol) in DMF (8 mL) at 00° C. underN₂, was added benzyl tert-butyl malonate (1.0 g, 4.0 mmol) in DMF (2mL). The mixture was stirred for 50 min after which 1-bromoundecane inDMF (2 mL) was added. After an additional hour of stirring the reactionwas allowed to warm to room temperature. The reaction was maintainedovernight. Et₂O (100 mL) and water (20 mL) were added to partition thereaction. The aqueous phase was extracted with Et₂O (100 mL), and thecombined organics dried over Na₂SO₄. The solvent was evaporated and theresidue purified by flash column (C18 12 g, 40-100% ACN/water+0.1% TFA)to yield the title compound as a colorless oil (1.14 g, 2.82 mmol, 71%):LCMS Method A Rt=1.58 min, M+Na 427.4; ¹H NMR (400 MHz, CHLOROFORM-d) δppm 0.84-0.96 (m, 3H) 1.28 (br. s, 12H) 1.31 (m, J=3.90 Hz, 6H) 1.41 (s,9H) 1.88 (q, J=7.38 Hz, 2H) 3.29 (t, J=7.58 Hz, 1H) 5.19 (q, J=12.27 Hz,2H) 7.30-7.42 (m, 5H).

Alternatively, alkylation of tert-butyl malonate can be carried outusing 1-iodoundecane (1.2 eq) in the presence of potassium carbonate (2eq) in DMF.

Intermediate 32: 1,11-Dibenzyl 11-tert-butylDocosane-1,11,11-tricarboxylate

The title compound was synthesized in a similar fashion as intermediate9 using intermediate 31 (650 mg, 1.61 mmol) as a starting material toyield a colorless oil (823 mg, 1.21 mmol, 75%): ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 0.84-0.94 (m, 3H) 1.12 (m, J=6.60 Hz, 4H) 1.19-1.33(m, 28H) 1.35 (s, 9H) 1.66 (quin, J=7.40 Hz, 2H) 1.85 (t, J=8.44 Hz, 4H)2.37 (t, J=7.52 Hz, 2H) 5.14 (s, 2H) 5.16 (s, 2H) 7.30-7.42 (m, 10H).

Intermediate 33:13-(Benzyloxy)-2-((benzyloxy)carbonyl)-13-oxo-2-undecyltridecanoic Acid

To a solution of intermediate 32 (200 mg, 0.295 mmol) in DCM (3 mL) wasadded TFA (0.6 mL), and the reaction stirred at room temperature for 3hrs. The solvent was evaporated and the residue purified by flash column(silica 12 g, 0-15% EtOAc/HEP) to yield the title compound (177 mg,0.284 mmol, 96%): ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.87-0.94 (m, 3H)0.94-1.05 (m, 2H) 1.19 (br. s., 14H) 1.23-1.37 (m, 16H) 1.65 (quin,J=7.40 Hz, 2H) 1.78-1.91 (m, 2H) 1.93-2.05 (m, 2H) 2.37 (t, J=7.52 Hz,2H) 5.14 (s, 2H) 5.27 (s, 2H) 7.31-7.44 (m, 10H).

Intermediate 34: 1,11-Dibenzyl 11-(2,5-dioxocyclopentyl)Docosane-1,11,11-tricarboxylate

The title compound was synthesized in a fashion similar to intermediate15 using intermediate 33 (177 mg, 0.284 mmol) as a starting material toyield a colorless oil (153 mg, 0.213 mmol, 75%): ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 0.86-0.93 (m, 3H) 1.12-1.21 (m, 2H) 1.21-1.37 (m,30H) 1.66 (quin, J=7.40 Hz, 2H) 1.89-2.07 (m, 4H) 2.37 (t, J=7.58 Hz,2H) 2.84 (br. s., 4H) 5.13 (s, 2H) 5.25 (s, 2H) 7.30-7.47 (m, 10H).

Intermediate 35

A solution of intermediate 34 (145 mg, 0.201 mmol) in THF (1.5 mL) andDCM (1.5 mL) was added to a vial charged with amino-PEG24-acid. DIPEA(88

L, 0.504 mmol) was added and the reaction agitated on a shaker plate for15 hrs. The solvent was evaporated and the residue purified bysupercritical fluid chromatography (Waters HILIC 20×150 mm; 15-25%MeOH/CO2) to yield intermediate 35 (151 mg, 0.086 mmol, 43%): LCMSMethod D Rt=1.30 min, [M+2H]⁺² 876.4; ¹H NMR (400 MHz, CHLOROFORM-d) δppm 0.86-0.93 (m, 3H) 0.93-1.04 (m, 2H) 1.19 (br. s., 15H) 1.23-1.37 (m,15H) 1.61-1.68 (m, 2H) 1.78 (td, J=12.44, 4.34 Hz, 2H) 1.92-2.05 (m, 2H)2.37 (t, J=7.58 Hz, 2H) 2.62 (t, J=6.05 Hz, 2H) 3.49 (dd, J=6.72, 2.32Hz, 2H) 3.52-3.59 (m, 2H) 3.59-3.73 (m, 92H) 3.80 (t, J=6.05 Hz, 2H)5.13 (s, 2H) 5.18 (s, 2H) 7.31-7.42 (m, 10H) 8.09 (t, J=5.26 Hz, 1H).

Intermediate 36

DCC (22 mg, 0.103 mmol) in DCM (0.265 mL) was added to a solution ofintermediate 35 (150 mg, 0.086 mmol) and N-hydroxysuccinimide in DCM(1.5 mL). The reaction was stirred for 1.5 hrs. AdditionalN-hydroxysuccinimide (10 mg) in THF (0.5 mL) and DCC (22 mg) in DCM(0.265 mL) was added and the reaction stirred overnight. The solvent wasevaporated and the residue purified by flash column (silica 12, 0-5%MeOH/DCM) to yield intermediate 36 (159 mg, quantitative) as a whitesolid: LCMS Method B Rt=1.55 min, [M+H₃O+H]⁺² 933.9.

Intermediate 37

To a solution of intermediate 36 (159 mg, 0.086 mmol) in THF (5 mL) wasadded a suspension of 10% Pd on carbon (4.6 mg, 4.3 μmol) in THF (1 mL).The reaction was placed under hydrogen and stirred for 40 min. More Pdon carbon (7 mg, 6.5 μmol) was added and the stirred another 1 hr underhydrogen. The reaction was passed through a membrane filter and thefiltrate evaporated. The residue was purified by HPLC (Sunfire C18 30×50mm, 45-70% ACN/water+0.1% TFA) to yield the title compound (83 mg, 0.047mmol, 54%): LCMS Method B Rt=1.03 min, [M+2H]+2 835.2; ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 0.84-0.94 (m, 3H) 1.17 (br. s., 2H) 1.21-1.39 (m,30H) 1.57-1.68 (m, 2H) 1.69-1.80 (m, 2H) 1.97-2.10 (m, 2H) 2.34 (t,J=7.21 Hz, 2H) 2.86 (s, 4H) 2.92 (t, J=6.48 Hz, 2H) 3.51-3.73 (m, 96H)3.87 (t, J=6.48 Hz, 2H) 7.45 (t, J=4.46 Hz, 1H)

Intermediate 38: 11-Bromoundec-1-yne

To a solution of 10-undecyn-1-ol (2.29 mL, 11.9 mmol) and carbontetrabromide (4.34 g, 13.1 mmol) in DCM (10 mL) under N2 at 0° C. wasadded triphenylphosphine (3.43 g, 13.1 mmol) portion-wise over 30 min.Upon completion of the addition the reaction was allowed to warm to roomtemperature. After 1.5 hr the reaction was poured into stirringcyclohexane (75 mL) and the precipitate collected. The solid was washedwith cyclohexane and the combined filtrates evaporated. The residue waspurified by flash column (silica 80 g, 0-10% EtOAc/HEP) to yield thetitle compound (1.75 g, 7.57 mmol, 64%): 1H NMR (400 MHz, CHLOROFORM-d)δ ppm 1.21-1.35 (m, 6H) 1.35-1.48 (m, 4H) 1.48-1.59 (m, 2H) 1.80-1.91(m, 2H) 1.94 (t, J=2.63 Hz, 1H) 2.19 (td, J=7.09, 2.69 Hz, 2H) 3.41 (t,J=6.85 Hz, 2H).

Intermediate 39: Di-tert-butyl 2-(undec-10-yn-1-yl)malonate

Di-tert-butyl malonate (800 mg, 3.70 mmol) was dissolved in DMF (9 mL)at 0° C. under N₂ and NaH (148 mg, 3.70 mmol) was added. The reactionwas stirred 30 minutes at 0° C. and intermediate 38 (770 mg, 3.33 mmol)was added slowly dropwise, resulting in a yellow solution. The reactionwas stirred at 0° C. for 2 hours then warmed to r.t. and stirred for 16hours. The mixture was taken up in EtOAc (75 mL) and washed with H₂O (25mL). The aqueous layer was extracted with EtOAc (75 mL) and the combinedorganic layers were dried over Na₂SO₄, filtered and concentrated. Themixture was purified twice via flash column (12 g silica cartridge,0-20% EtOAc/heptanes) and fractions were concentrated to yield 162.1 mgof the desired product as a colorless oil (12%). LCMS (Waters AcquityUPLC BEH C18, 130 Å, 1.7 μm, 2.1 mm×50 mm, 50° C., Solvent Name A:Water+0.1% Formic Acid, Solvent Name B: Acetonitrile+0.1% Formic Acid,98% B over 2.20 min): R_(t)=1.37 min, MS [M+H] observed: 366.0,calculated: 366.535. ¹H NMR (400 MHz, Chloroform-d) δ ppm 1.29 (s, 10H)1.47 (s, 18H) 1.52 (dd, J=14.78, 7.20 Hz, 3H) 1.48 (d, J=1.26 Hz, 1H)1.75-1.83 (m, 2H) 1.94 (t, J=2.65 Hz, 1H) 2.18 (td, J=7.14, 2.65 Hz, 2H)3.11 (t, J=7.58 Hz, 1H).

Intermediate 40: 11,11-di-tert-butyl 1-ethyldocos-21-yne-1,11,11-tricarboxylate

Intermediate 39 (162.1 mg, 0.442 mmol) was dissolved in DMF (2 mL) at 0°C. and NaH (21.23 mg, 0.531 mmol) was added. The reaction stirred at 0°C. for 15 minutes and ethyl 11-bromoundecanoate (143 mg, 0.486 mmol) wasadded slowly dropwise. The reaction was warmed to r.t. and stirred for16 hours. The mixture was diluted with EtOAc (40 mL) and washed oncewith H₂O (20 mL). The aqueous layer was extracted once with EtOAc (40mL) and the organic layers were combined, dried over Na₂SO₄, filteredand concentrated to give a clear, yellow oil. The sample was dissolvedin 1 mL DCM and purified via flash column (12 g silica column, 0-20%EtOAc/heptane, 15 min). The fractions were combined and concentrated togive 90.1 mg of the desired product (35%). ¹H NMR (400 MHz,Chloroform-d) δ ppm 1.28 (br. s., 24H) 1.45 (s, 18H) 1.48 (s, 3H) 1.53(d, J=7.58 Hz, 3H) 1.51 (s, 1H) 1.64 (br. s., 1H) 1.61 (d, J=7.33 Hz,1H) 1.77 (d, J=16.93 Hz, 2H) 1.74-1.80 (m, 2H) 1.94 (t, J=2.65 Hz, 1H)2.18 (td, J=7.07, 2.53 Hz, 2H) 2.29 (t, J=7.58 Hz, 2H) 4.13 (q, J=7.24Hz, 2H).

Intermediate 41: 12,12-bis(tert-butoxycarbonyl)tricos-22-ynoic Acid

To a solution of intermediate 40 (21.7 mg, 0.037 mmol) in ^(t)BuOH (1mL) was added a solution of KOtBu (114 mg, 1.012 mmol) in ^(t)BuOH (2mL) at 30° C. under N₂. The mixture was stirred at r.t. and monitored byTLC (1:1 EtOAc/hexanes, KMnO₄, reflux). The starting material wasconsumed after 3 hours and the reaction mixture was quenched with 1 MHCl (20 mL) and extracted twice with EtOAc (25 mL). The organic layerswere combined, dried over Na₂SO₄, filtered and concentrated to a clear,colorless oil (18 mg, 87%). The material was carried on to the next stepwithout further purification. ¹H NMR (400 MHz, Chloroform-d) δ ppm 1.27(br. s., 22H) 1.44 (br. s., 18H) 1.48 (s, 3H) 1.52 (s, 3H) 1.62 (br. s.,2H) 1.77 (br. s., 4H) 1.94 (br. s., 1H) 2.18 (s, 2H) 2.35 (s, 2H).

Intermediate 42: Docos-21-yne-1,11,11-tricarboxylic Acid

TFA (2 mL) was added to intermediate 41 (12 mg, 0.022 mmol) and thereaction stirred at r.t. for 1 hour. The mixture was diluted with DCM(10 mL) and concentrated twice to give a brown oil. The material wastaken up in EtOAc (10 mL) and washed with H₂O (20 mL). The organic layerwas dried over Na₂SO₄, filtered and concentrated to a brown oil. Thecrude material was dissolved in 1 mL MeOH and purified via MS-triggeredHPLC (Sunfire 30×50 mm 5 um column ACN/H2O w/0.1% TFA 75 ml/min, 1.5 mlinjection, 45-70% ACN over 3.5 min): R_(t)=3.42 min; MS [M+H+Na]observed: 461.00, calculated: 461.597. Fractions were pooled andlyophilized to give 5.3 mg of title compound in 56% yield.

Intermediate 43:2-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)-2-(undec-10-yn-1-yl)tridecanedioicAcid

To a solution of intermediate 42 (5.3 mg, 0.012 mmol) in THF (0.5 mL)was added N-hydroxy succinimide (1.53 mg, 0.013 mmol). A solution of DCC(2.493 mg, 0.012 mmol) in THF (0.5 mL) was added and the mixture wasstirred at r.t. under N₂ for 4 hours. Complete conversion of startingmaterial was observed by LCMS. The mixture was concentrated and taken onto the next step without further purification. LCMS (Sunfire C18 3.5 μm3.0×30 mm, 40° C., Acidic Eluent A: Water+0.05% Trifluoroacetic Acid,Basic Eluent A: Water+5 mM Ammonium Hydroxide, Eluent B: ACN, 5-95% over2 min): R_(t)=1.72 min; MS [M+H+Na] observed: 558.0, calculated: 558.67.

Intermediate 44

To a solution of intermediate 43 (3.2 mg, 5.97 μmol) in DCM (0.5 mL) wasadded a solution of azido-dPEG23-amine (Quanta Biodesign, 7.88 mg, 7.17μmol) in DCM (0.5 mL) and DIPEA (2.09 μL, 0.012 mmol) and the mixturewas stirred at r.t. for 16 hours at which point conversion of startingmaterial was observed by LCMS. The reaction mixture was concentrated anddissolved in 1 mL MeOH and purified by MS-triggered HPLC (Sunfire 30×50mm 5 um column ACN/H2O w/0.1% TFA 75 ml/min, 1.5 ml injection, 55-80%ACN 5 min gradient, R_(t)=1.92 min) and the fractions were pooled andlyophilized to give 1.7 mg of the title compound in 19% yield. LCMS(Acquity BEH 1.7 μm 2.1×50 mm−50° C., Solvent Name A: Water+0.1% FormicAcid, Solvent Name B: Acetonitrile+0.1% Formic Acid, 98% B over 2.20min): R_(t)=1.89 min; MS [M+H/2] observed: 760.0, calculated: 759.5.

Intermediate 45: docos-21-ene-1,11,11-tricarboxylic Acid

Intermediate 45 is prepared following the procedure for intermediate39-42 substituting 11-bromo-dec-1-ene for 11-bromo-dec-1-yne.

Intermediate 46:2-(((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)-2-(undec-10-en-1-yl)tridecanedioicAcid

DCC (187 mg, 0.908 mmol) in DCM (2 mL) was added to a solution ofN-hydroxysuccinimide (99 mg, 0.862 mmol) anddocos-21-ene-1,11,11-tricarboxylic acid (Intermediate 45: 400 mg, 0.908mmol) in DCM (7 mL) and THF (0.7 mL). The reaction was stirred overnightbefore the solvent was evaporated. The residue was purified by HPLC(Sunfire C18 30×50 mm; 55-80% ACN/water+0.1% TFA) to yield the titlecompound (155 mg, 0.288 mmol, 32%): by LCMS Method C Rt=1.51 min, M+H538.3; ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.16-1.46 (m, 28H) 1.60-1.87(m, 3H) 1.91-2.17 (m, 5H) 2.38 (t, J=7.03 Hz, 2H) 2.86 (br. s., 4H) 3.68(dd, J=11.25, 7.34 Hz, 1H) 3.78 (dd, J=11.31, 5.20 Hz, 1H) 3.99-4.10 (m,1H).

Intermediates 47 and 47A

Azido-dPEG23-amine (Quanta Biodesign: 164 mg, 0.149 mmol) andintermediate 46 (80 mg, 0.149 mmol) were dissolved in THF (2.5 mL).DIPEA (39 μL, 0.233 mmol) was added and the reaction agitated overnight.The solvent was evaporated and the residue purified by HPLC (Sunfire C1830×50 mm; 45-70% ACN/water+0.1% TFA) to yield compounds 47 (97 mg, 0.061mmol, 41%) and 47A (32 mg, 0.021 mmol, 14%): LCMS Method C Rt=1.35 min,[M+2H]⁺² 761.9; ¹H NMR (400 MHz, ACETONITRILE-d₃) δ ppm 1.05-1.18 (m,3H) 1.19-1.32 (m, 20H) 1.36 (t, J=7.15 Hz, 1H) 1.48-1.59 (m, 2H)1.65-1.75 (m, 2H) 2.01-2.06 (m, 2H) 2.25 (t, J=7.46 Hz, 2H) 3.33-3.39(m, 2H) 3.39-3.44 (m, 2H) 3.50-3.67 (m, 98H) 4.84-4.95 (m, 1H) 4.95-5.06(m, 1H) 5.83 (ddt, J=17.07, 10.29, 6.68, 6.68 Hz, 1H) 7.31 (t, J=5.44Hz, 1H); LCMS method C Rt=1.50 min, [M+2H]⁺² 739.9; ¹H NMR (400 MHz,ACETONITRILE-d₃) δ ppm 1.16-1.42 (m, 30H) 1.42-1.63 (m, 5H) 2.00-2.07(m, 2H) 2.22-2.28 (m, 2H) 2.40-2.52 (m, 2H) 3.25-3.33 (m, 2H) 3.33-3.42(m, 2H) 3.42-3.50 (m, 2H) 3.50-3.68 (m, 88H) 4.86-5.06 (m, 2H) 5.83(ddt, J=17.04, 10.26, 6.71, 6.71 Hz, 1H) 6.40-6.74 (m, 1H).

Intermediate 48: 2-Undecylmalonic Acid

Using intermediate 22 (290 mg, 0.661 mmol), the title compound (185 mg,quantitative) was synthesized in a similar fashion as intermediate 19:LCMS Method B LCMS Rt=0.82 min, M−H 257.3

Intermediate 49: 2-(((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)tridecanoicAcid

DCC (122 mg, 0.592 mmol) was added to a solution of intermediate 48 (170mg, 0.658 mmol) and N-hydroxysuccinimide (68 mg, 0.592 mmol) in DCM (6mL) and THF (0.5 mL). The reaction was stirred for 16 hrs before moreDCC (30 mg, 0.145 mmol) in DCM (0.5 mL) was added. The reaction wasstirred for a further 2 days. The solvent was evaporated and the residuepurified by HPLC (Sunfire C18 30×50 mm, 45-70% ACN/water+0.1% TFA) toyield the title compound as a white powder (46 mg, 0.129 mg, 20%): LCMSMethod B Rt=0.94 min, M+H 356.3; ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm0.89 (t, J=7.00 Hz, 3H) 1.20-1.40 (m, 16H) 1.43-1.55 (m, 2H) 1.99-2.14(m, 2H) 2.86 (s, 4H) 3.71 (t, J=7.46 Hz, 1H).

Intermediate 50 and 50A

The title compounds were synthesized in a fashion similar to 50 and 50Afrom intermediate 49 (30 mg, 0.084 mmol) yielding intermediate 50 (18mg, 0.013 mmol, 16%) and intermediate 50A (5 mg, 4 mol, 5%): LCMS MethodB Rt=0.85 min, M+H 1340.3; ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm0.82-0.98 (m, 3H) 1.20-1.36 (m, 16H) 1.36-1.51 (m, 2H) 1.83-2.02 (m, 1H)2.11-2.27 (m, 1H) 2.33 (dd, J=11.80, 4.22 Hz, 1H) 3.41 (t, J=5.14 Hz,3H) 3.49 (d, J=5.01 Hz, 1H) 3.56-3.79 (m, 92H); LCMS Method B Rt=0.96min, M+H 1296.3.

Intermediates 51-57: Mutant of GDF15 Protein

Expression of Human GDF-15 Proteins in E. coli Cells

-   -   E. coli strains BL21 (DE3) Gold (Stratagene) and Rosetta (DE3)        pLysS cells (Novagen) were transformed with constructs 51 to 56        and construct MAHA-(200-308)-hGDF15 respectively, cloned into        pET26b vectors. Transformed cells were grown under antibiotic        selection first in 3 ml and then in 50 ml Luria Broth        (Bacto-Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5/L, glucose 6        g/L) until an OD600 of 1.5 was reached. The pre-cultures were        used to inoculate two 1-L fermenters filled with Terrific Broth        medium (NH4SO4 1.2 g/L, H2PO4 0.041 g/L, K2HPO4 0.052 g/L,        Bacto-Tryptone 12 g/L, Yeast Extract 24 g/L). The cultures were        induced by automatic addition of 1 mM        isopropyl-beta-D-thiogalactopyranoside (IPTG) when pH increased        above 7.1. Other fermentation parameters were: temp=37° C.; pH        7.0+/−0.2 adjusted by addition of 2N NaOH/H₂SO₄; pO₂>30% with        cascades of stirrer speed, air flow and oxygen addition. Five        hours post induction the cultures were cooled to 10° C. and        cells were harvested by centrifugation.        Purification and Refolding of GDF15 Variants

a) Inclusion Bodies

-   -   Recombinant coli pellets expressing the protein of interest were        resuspended (5% w/v) in 50 mM NaH₂PO₄/150 mM NaCl/5 mM        benzamidine.HCl/5 mM DTT, pH 8.0 at 4° C., homogenized and lysed        by 2 passages through a French press (800 and 80 bar). Inclusion        bodies (IBs) were isolated by centrifugation at 12′000 rpm for        60 min at 4° C.

b) Purification of Crude Unfolded Protein

-   -   IBs were solubilized (5% w/v) in 6 M guanidine/100 mM NaH₂PO₄/10        mM Tris/20 mM beta-mercaptoethanol, pH 8.0 and stirred for 2        hours at room temperature. Debris was removed by centrifugation        at 12′000 rpm. The solubilized IBs were further purified on        Ni-NTA-Superflow (the construct without His tag binds as well to        this resin due to the high histidine content). After base-line        washing with 6 M guanidine/100 mM NaH₂PO₄/10 mM Tris/5 mM        beta-mercaptoethanol, pH 8.0, bound material was eluted with the        same buffer adjusted to pH 4.5. The eluate was adjusted to pH        8.0, 100 mM DTT was added and the solution stirred over night at        4° C. The pH was then adjusted to 2 by addition of        trifluoroacetic acid (TFA, 10% stock in water) and the solution        further diluted 1:1 with 0.1% TFA in water. The crude protein        solution was further purified by RP-HPLC (Poros) using a        gradient of 0-50% acetonitrile in 50 min. The GDF-15 containing        fractions were pooled and lyophilized.

c) Protein Folding

-   -   Method 1: Lyophilized material was dissolved at 2 mg/ml in 100        mM acetic acid, diluted 15-20 folds in folding buffer (100 mM        CHES/1 M NaCl/30 mM CHAPS/5 mM GSH/0.5 mM GSSG/20% DMSO, pH 9.5,        4° C.) and the solution gently stirred during 3 days at 4° C.        After 3 days 3 volumes of 100 mM acetic acid was added and the        solution concentrated by ultrafiltration (5 kDa cut-off) to        about 100-200 ml, diluted 10 fold with 100 mM acetic acid and        re-concentrated. The refolded material was further purified by        preparative RP-HPLC on a Vydac C4 column run at 50° C. (buffer        A: 0.1% TFA in water; buffer B: 0.05% TFA in acetonitrile).        After loading the column was washed with 15% buffer B and eluted        with a gradient of 15% B to 65% B in 50 min. Collected fractions        containing the protein of interest were diluted with an equal        volume of buffer A and lyophilized. Refolding yields were about        25% for both proteins.    -   Method 2: Protocol followed as in method 1 with folding buffer:        100 mM CHES, pH 9.4, 0.9 M arginine, 0.5 M NaCl, 1 mM EDTA, 2.5        mM GSH, 1 mM GSSG (final concentration).

The following GDF15 mutants were prepared according to above procedure:

Intermediate 51: M-(His)₆-hGDF15

(SEQ ID NO: 1) MHHHH HHAR NGDHC PLGPG RCCRL HTVRA SLEDL GWADW VLSPREVQVT MCIGA CPSQF RAANM HAQIK TSLHR LKPDT VPAPC CVPAS YNPMV LIQKT DTGVSLQTYD DLLAK DCHCI

LCMS: Calculated mass: 26462 Observed Mass: 26464

Intermediate 52: M-(His)₆-M-hGDF15

(SEQ ID NO: 2) MHHHHHHMARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKT DTGVSLQTYDDLLAKDCHCI

LCMS: Calculated mass: 26724 Observed Mass: 26725

Intermediate 53: His-dGDF15

(SEQ ID NO: 3) MHHHHHHAHARDGCPLGEGRCCRLQSLRASLQDLGWANWVVAPRELDVRMCVGACPSQFRSANTHAQMQARLHGLNPDAAPAPCCVPASYEPVVLMHQDS DGRVSLTPFDDLVAKDCHCV

LCMS: Calculated mass (dimer): 26368 Observed Mass: 26363

Intermediate 54: MH-(199-308)-hGDF15

(SEQ ID NO: 4) MHNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQT YDDLLAKDCHCI

LCMS: Calculated mass: 24636 Observed Mass: 24638

Intermediate 55: AH-(199-308)-hGDF15

(SEQ ID NO: 5) AHNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQT YDDLLAKDCHCI

Step 1: preparation of construct M-His₆-TEV(ENLYFQ/A)-H-hsGDF15aa199-308 The construct M-His6-TEV(ENLYFQ/A)-H-hsGDF15 aa199-308 wasprepared according to the above procedure (steps a, b and c).

Step 2: TEV cleavage of the protein from step 1

The lyophilized protein was solubilized in water to a finalconcentration of 1.75 mg/ml. The protein was unfolded again by diluting1:1 in 6M Guan/50 mM Tris, pH 8.0+50 mM DTT, and stirred at RT for 1 h.The material was re-purified by preparative RP-HPLC on a Vydac C4 columnand lyophilized. 26 mg of lyophilisate were solubilized in 26 ml 50 mMTris/3M UREA, pH 7.5+3000 Units AcTEV Protease (Invitrogen, 12575-023)and incubated for 4 days. The pH was then adjusted to pH 2.0 by additionof trifluoroacetic acid (TFA, 10% stock in water) and the solutionfurther diluted to 150 ml with 0.1% TFA in water. After filtrationthrough a 0.22 um membrane the material was again purified bypreparative RP-HPLC on a Vydac C4 column to isolate successfully cleavedprotein. Fractions were collected manually; target protein-containingfractions were isolated and lyophilized. The cleaved GDF15 protein wasthen refolded and refolded protein purified as described above.

LCMS: Calculated mass (dimer): 24516 Observed Mass: 24518

The following GDF15 mutant can be prepared according to the aboveprocedure:

Intermediate 56: MHA-(200-308)-hGDF15

(SEQ ID NO: 6) MHAGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQT YDDLLAKDCHCI

LCMS: Calculated mass (dimer): 24752

The following GDF15 mutant was prepared according to the aboveprocedure:

Intermediate 57: AHA-(200-308)-hGDF15

(SEQ ID NO: 7) AHAGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQT YDDLLAKDCHCI

LCMS: Calculated mass (dimer): 24430: Observed mass (dimer): 24432

Intermediate 58: His-hGDF15 BCN Conjugate

His-hGDF15

Seq: (SEQ. ID. NO: 2) MHHHHHHMARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKT DTGVSLQTYDDLLAKDCHCI

A stock solution of His-hGDF15 (Intermediate 52: 0.6 mL, 4.8 mg/mL) wasdiluted to 0.5 mg/mL with 30 mM NaOAc pH 4.5 buffer (5.2 mL). A 10 mg/mLstock solution of (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl(2,5-dioxopyrrolidin-1-yl) carbonate (NHS-BCN) in DMSO (251 μL) wasslowly added and the reaction placed on a shaker plate at 24° C. for 21hrs. The reaction was diluted to 30 mL with 30 mM NaOAc pH 4.5 bufferand concentrated to 2 mL using a 10 kDa MWCO ultrafiltration cartridge(repeat 4×) to yield 2.5 mL of concentrate. Based on A₂₈₀ (ε=29090M⁻¹cm⁻¹, 26730 g/mol) the concentrate was 0.93 mg/mL (2.33 mg, 80%): LCMSQT2_15-60 kDa_15 min_polar (method E). The resulting solution wasanalyzed by MALDI to indicate major conjugation to be +1 and +2(N-terminus labeling of monomer and dimer)

% TIC Degree of Labelling Calculated Observed (MS+) Intensity GDF1526726 26726 26 GDF15 + 1BCN 26903 26904 43 GDF15 + 2BCN 27080 27080 23GDF15 + 3BCN 27257 27256 9

His-hGDF15 +1BCN (bicyclo[6.1.0]non-4-ynyl) corresponds to a reaction atthe N-terminus amino functionality on the one molecule of the GDF15homodimer.

His-hGDF15 +2BCN corresponds to a reaction at the N-terminus aminofunctionality on both monomeric units of the GDF15 homodimer.

His-hGDF15 +3BCN corresponds to a non-selective reaction at some othersite of the GDF15 homodimer.

Intermediate 59: His-hGDF15 BCN Conjugate

His-hGDF15 Seq:

(SEQ. ID. NO: 1) MHHHH HHAR NGDHC PLGPG RCCRL HTVRA SLEDL GWADW VLSPREVQVT MCIGA CPSQF RAANM HAQIK TSLHR LKPDT VPAPC CVPAS YNPMV LIQKT DTGVSLQTYD DLLAK DCHCI

A stock solution of His-hGDF15 (Intermediate 51: 7.04 mL, 1.42 mg/mL)was diluted to 0.5 mg/mL with 30 mM NaOAc pH 4.5 buffer (12.95 mL). A 10mg/mL stock solution of (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl(2,5-dioxopyrrolidin-1-yl) carbonate (NHS-BCN) in DMSO (0.88 mL) wasslowly added and the reaction placed on a shaker plate at 24° C. for 24hrs. An additional portion of the NHS-BCN stock solution (176 L) wasadded, and the reaction maintained at 24° C. for 24 hrs. The reactionwas diluted to 60 mL with 30 mM NaOAc pH 4.5 buffer and concentrated to4 mL using a 10 kDa MWCO ultrafiltration cartridge (repeat 4×) to yield4.1 mL of concentrate. Based on A₂₈₀ (s=29090M⁻¹ cm⁻¹, 26700 g/mol) theconcentrate was 2.19 mg/mL (8.98 mg, 89%): LCMS QT2_15-60 kDa_15min_polar (Method E)

% TIC (MS+) Degree of Labelling Calculated Observed Intensity GDF1526468 26464 33 GDF15 + 1BCN 26645 26640 34 GDF15 + 2BCN 26822 26817 21GDF15 + 3BCN 26999 26993 3

His-hGDF15 +1BCN (bicyclo[6.1.0]non-4-ynyl) corresponds to a reaction atthe N-terminus amino functionality on the one molecule of the GDF15homodimer.

His-hGDF15 +2BCN corresponds to a reaction at the N-terminus aminofunctionality on both monomeric units of the GDF15 homodimer.

His-hGDF15 +3BCN corresponds to a non-selective reaction at some othersite of the GDF15 homodimer.

Intermediate 60: His-dog-GDF15-BCN

100 uL His-dGDF15 (0.68 mg/mL), 100 uL pH 4.5 buffer, 4 uL of a 10 mg/mLBCN-NHS solution was combined and incubated at rt for two days. Theresulting mixture was washed with Amicon 10k 4 times to give 200 uLsolution, which was used in next step. The product was carried on ascrude for further conversion to conjugate.

EXAMPLES OF THE INVENTION

General Procedure for his-hGDF15+Fatty Acid-PEG-N₃ Click.

BCN labelled GDF15 was diluted to 0.5 mg/mL in 30 mM NaOAc pH 4.5 bufferwhile a 10 mg/mL solution of FA-PEG-N₃ (fatty acid-PEG23-azide) in waterwas prepared. To the GDF15 solution was added 10 eq of FA-PEG-N₃, andthe reaction was placed on a shaker plate at 24° C. overnight. Reactionprogress was monitored by LCMS (QTOF method 15-60 kDa_15 min_polar) andadditional FA-PEG-N₃ was added, if necessary up to 50 eq, until nounreacted BCN labelled GDF15 was observed. The reaction was then diluted5-10× with 30 mM NaOAc pH 4 buffer and the buffer exchanged with freshbuffer using a 10 kDa MWCO ultrafiltration cartridge (4 cycles ofconcentration followed by dilution). The sample was concentrated to ˜1mg/mL as measured by A₂₈₀, recoveries were quantitative to 34%. Finalconjugates were analyzed by LCMS (QTOF method 15-60 kDa_15 min_polar) orMaldi.

Example 1: His-hGDF15 BCN (I-59) Conjugated to Intermediate 21

Degree of Labelling Calculated Observed % AUC @ 280 nm His-hGDF15 2646826466 18 His-hGDF15 + 1FA 28198 28192 36 His-hGDF15 + 2FA 29928 29926 35His-hGDF15 + 3FA 31658 31654 11

His-hGDF15 +1FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on the one molecule (one monomeric unit) of theGDF15 homodimer.

His-hGDF15 +2FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on both monomeric units of the GDF15 homodimer.

His-hGDF15 +3FA (Fatty acid) corresponds to a non-selective reaction atsome other site of the GDF15 homodimer.

Example 2: His-hGDF15 BCN (I-59) Conjugated to Intermediate 44

Degree of Labelling Calculated Observed % AUC @ 280 nm His-hGDF15 2646426464 38 His-hGDF15 + 1FA 28162 28162 33 His-hGDF15 + 2FA 29860 29860 21His-hGDF15 + 3FA 31558 31558 9

His-hGDF15 +1FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on the one molecule (one monomeric unit) of theGDF15 homodimer.

His-hGDF15 +2FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on both monomeric units of the GDF15 homodimer.

His-hGDF15 +3FA (Fatty acid) corresponds to a non-selective reaction atsome other site of the GDF15 homodimer.

Example 3: His-hGDF15 BCN (I-59) Conjugated to Intermediate 29A

Degree of Labelling Calculated Observed % AUC @ 280 nm His-hGDF15 2646426466 50 His-hGDF15 + 1FA 28124 28120 28 His-hGDF15 + 2FA 29780 29776 16His-hGDF15 + 3FA 31436 31432 7

His-hGDF15 +1FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on the one molecule (one monomeric unit) of theGDF15 homodimer.

His-hGDF15 +2FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on both monomeric units of the GDF15 homodimer.

His-hGDF15 +3FA (Fatty acid) corresponds to a non-selective reaction atsome other site of the GDF15 homodimer.

Example 4: His-hGDF15 BCN (Intermediate 58) Conjugated Intermediate 24

Degree of Labelling Calculated Observed % AUC @ 280 nm His-hGDF15 2672626728 27 His-hGDF15 + 1FA 28396 28398 42 His-hGDF15 + 2FA 30066 30068 24His-hGDF15 + 3FA 31736 31738 7

His-hGDF15 +1FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on the one molecule (one monomeric unit) of theGDF15 homodimer.

His-hGDF15 +2FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on both monomeric units of the GDF15 homodimer.

His-hGDF15 +3FA (Fatty acid) corresponds to a non-selective reaction atsome other site of the GDF15 homodimer.

Example 5: His-hGDF15 BCN (I-58) Conjugated to Intermediate 29

Degree of Labelling Calculated Observed % AUC @ 280 nm His-hGDF15 2672626728 30 His-hGDF15 + 1FA 28425 28426 36 His-hGDF15 + 2FA 30125 30126 23His-hGDF15 + 3FA 31825 31740 12

His-hGDF15 +1FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on the one molecule of the GDF15 homodimer.

His-hGDF15 +2FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on both monomeric units of the GDF15 homodimer.

His-hGDF15 +3FA (Fatty acid) corresponds to a non-selective reaction atsome other site of the GDF15 homodimer.

Example 6: His-hGDF15 BCN (I-58) Conjugated to Intermediate 55

Degree of Labelling Calculated Observed % AUC @ 280 nm His-hGDF15 2672626728 28 His-hGDF15 + 1FA 28242 28243 36 His-hGDF15 + 2FA 29758 29759 28His-hGDF15 + 3FA 31274 31275 11

His-hGDF15 +1FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on the one molecule of the GDF15 homodimer.

His-hGDF15 +2FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on both monomeric units of the GDF15 homodimer.

His-hGDF15 +3FA (Fatty acid) corresponds to a non-selective reaction atsome other site of the GDF15 homodimer.

Example 7: His-hGDF15 BCN (I-58) Conjugated to Intermediate 6A

Degree of Labelling Calculated Observed % AUC @ 280 nm His-hGDF15 2672626728 28 His-hGDF15 + 1FA 28382 28382 42 His-hGDF15 + 2FA 30038 30040 29His-hGDF15 + 3FA 31916 n/a n/a

His-hGDF15 +1FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on the one molecule (one monomeric unit) of theGDF15 homodimer.

His-hGDF15 +2FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on both monomeric units of the GDF15 homodimer.

His-hGDF15 +3FA (Fatty acid) corresponds to a non-selective reaction atsome other site of the GDF15 homodimer.

Example 9: His-hGDF15 BCN (I-58) Conjugated to Intermediate 30

Degree of Labelling Calculated Observed % AUC @ 280 nm His-hGDF15 2672626728 21 His-hGDF15 + 1FA 28456 28456 47 His-hGDF15 + 2FA 30186 30188 32His-hGDF15 + 3FA 31916 n/a n/a

His-hGDF15 +1FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on the one molecule (monomeric unit) of the GDF15homodimer.

His-hGDF15 +2FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on both molecules (monomeric units) of the GDF15homodimer.

His-hGDF15 +3FA (Fatty acid) corresponds to a non-selective reaction atsome other site of the GDF15 homodimer.

Example 10: His-hGDF15 BCN (I-58) Conjugated to Intermediate 12

Degree of Labelling Calculated Observed % AUC @ 280 nm His-hGDF15 2672626729 17 His-hGDF15 + 1FA 28442 28445 37 His-hGDF15 + 2FA 30158 30158 32His-hGDF15 + 3FA 31874 31877 13

His-hGDF15 +1FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on the one molecule (one monomeric unit) of theGDF15 homodimer.

His-hGDF15 +2FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on both molecules (monomeric units) of the GDF15homodimer.

His-hGDF15 +3FA (Fatty acid) corresponds to a non-selective reaction atsome other site of the GDF15 homodimer.

Example 13: His-hGDF15 BCN (I-59) Conjugated to Intermediate 6

Degree of Labelling Calculated Observed % AUC @ 280 nm His-hGDF15 2646826464 28 His-hGDF15 + 1FA 28168 28164 42 His-hGDF15 + 2FA 29868 29864 21His-hGDF15 + 3FA 31568 31564 10

His-hGDF15 +1FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on the one molecule (one monomeric unit) of theGDF15 homodimer.

His-hGDF15 +2FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on both molecules (both monomeric units) of theGDF15 homodimer.

His-hGDF15 +3FA (Fatty acid) corresponds to a non-selective reaction atsome other site of the GDF15 homodimer.

Example 14: His-hGDF15 BCN (I-58) Conjugated to Intermediate 17

Degree of Loading Calculated Observed % AUC @ 280 nm His-hGDF15 2672626728 18 His-hGDF15 + 1FA 28413 28414 34 His-hGDF15 + 2FA 30100 30054 35His-hGDF15 + 3FA 31787 31726 13

His-hGDF15 +1FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on the one molecule (one monomeric unit) of theGDF15 homodimer.

His-hGDF15 +2FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on both molecules (both monomeric units) of theGDF15 homodimer.

His-hGDF15 +3FA (Fatty acid) corresponds to a non-selective reaction atsome other site of the GDF15 homodimer.

Example 15 Step 1

To a solution of 2,2,13,13-tetramethyltetradecanedioic acid (AldrichCPR, order number PH002322) (100 mg, 0.318 mmol) andN-hydroxysuccinimide (40.3 mg, 0.35 mmol) in THF (5 mL) was added asolution of DCC (65.6 mg, 0.318 mmol) in THF (5 mL), and the mixture wasstirred at r.t. overnight. Partial conversion to the desired product wasobserved by LC-MS analysis. The mixture was filtered, and the filtratewas concentrated. The residue was re-dissolved in DCM (40 mL), andwashed with water, dried over Na2SO4, and purified by silicachromatography eluting with a heptane/EtOAc/DCC (10:1:1) to give amixture. The mixture was further purified by MS triggered acid HPLC[(55-80% ACN 3.5 min gradient): rt=2.48 min, mass calculated: 314.46mass observed: 314.00] to give to give clean product (50 mg, 38.2%yield) and to recover starting material.

Step 2

To a solution of NHS-2,2,13,13-tetramethyltetradecanedioic acid (10 mg,0.024 mmol) in DCM (3 mL) was added azido-dPEG3-amine (10 mg, 0.049mmol) and DIPEA (9 uL, 0.049 mmol), and the mixture was stirred at r.t.for 1 h. The mixture was concentrated, re-dissolved in MeOH (3 mL) andpurified by MS-triggered HPLC (55-80% ACN 3.5 min gradient rt=2.35, massexpected: 514.70 mass observed: 514.40) to give 7 mg clean product in58% yield.

Step 3

To a solution of 100 μL BCN-dGDF15 (I-60: 0.68 mg/mL in pH 4.5 buffer)was added pH 4.5 buffer (100 μL) and azide (6 μL in DMSO, 10 mg/mL), andthe mixture was incubated at r.t. overnight. The mixture was washed byAmicon 10k 4 times. The resulting solution was analyzed by MALDI toindicate major conjugation to +1 and +2. Maldi: Calculated mass: 26546Observed mass: 26483; Calculated mass: 27060 Observed mass: 27128;Calculated mass: 27574 Observed mass: 27789.

Example 16: His-hGDF15 BCN (I-58) Conjugated to Intermediate 44

Degree of Labelling Calculated Observed % AUC @ 280 nm His-hGDF15 2672626728 45 His-hGDF15-BCN 26902 26904 21 His-hGDF15 + 1FA 28422 28360 25His-hGDF15 + 2FA 29868 30012 9

His-hGDF15 +1FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on the one molecule (one monomeric unit) of theGDF15 homodimer.

His-hGDF15 +2FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on both molecules (both monomeric units) of theGDF15 homodimer.

Example 17: His-hGDF15-BCN Conjugated to Intermediate 52

To a solution of 3 mL cyclooctyne GDF15 (I-58: 0.46 mg/mL, 0.051 umol)in 7 mL of pH 4 sodium acetate buffer was added fatty acid peg azide (15uL in 35 mg/mL DMSO, 0.36 umol), and the mixture was incubated at r.t.overnight. Complete conversion was observed by MALDI analysis. Productpurified by amicon filtration 10 kD washing three times to give 4.3 mlof 0.29 mg/ml desired product in 90% yield. Maldi: cyclooctyne sm ˜5%expected mass: 26902 mass observed: 26997; +1 fatty acid ˜40% expectedmass: 28421 observed mass: 28525; +2 fatty acids ˜50% expected mass:29940 observed mass: 30191 +3 fatty acids 5% expected mass: 31459observed mass: 31874.

Example 18: MH-(199-308)GDF15 (I-54) Conjugated to Intermediate 37

MH-(199-308)-GDF15 (Intermediate 54: 0.393 mL, 0.028 μmol, 1.78 mg/mL)was added to 1.5 ml of 30 mM sodium acetate buffer NHS fatty acid (474ug, 0.284 umol, 10 mg/ml) was added to solution. After 5 hours, reactionwas complete according to MALDI. Products were purified by washing 5times using amicon ultrafiltration 10 kD to give 575 ug of conjugate in73% yield. MALDI: sm (18%), expected mass: 24638 observed mass: 24735;+1 fatty acid (38%) expected mass: 26192 observed mass: 26268; +2 fattyacid (40%) expected mass: 27746 observed mass: 27798; +3 fatty acid (4%)expected mass: 29300 observed mass: 29333.

Example 19A: His-hGDF15 (I-59) Conjugated to Intermediate 37

His-GDF15 (0.493 ml, 0.026 μmol, 1.42 mg/ml) was added to 1.5 ml of 30mM sodium acetate pH=4 buffer nhs fatty acid (0.221 mg, 0.132 umol, 10mg/mL) was added to the solution. Overnight the reaction was notcomplete so 2.5 more equivalents of fatty acid NHS (0.110 mg, 0.066umol, 10 mg/mL) were added and after 5 hrs Maldi showed +2 conjugate asmajor product. Product was purified by washing 5 times using amiconultrafiltration 10 kD to give 565 ug of conjugate in 76% yield. MALDI:sm (18%), expected mass: 26468 observed mass: 26553; +1 fatty acid (38%)expected mass: 28022 observed mass: 28099; +2 fatty acid (40%) expectedmass: 29576 observed mass: 29649; +3 fatty acid (4%) expected mass:31130 observed mass: 31201.

Example 19B: AHA-hGDF15 Conjugated with Intermediate 37

A 10 mg/mL solution of Intermediate 37 in molecular biology grade waterwas prepared. To AHA-hGDF15 (intermediate 57, 6.67 mg/mL in 30 mM NaOAcpH 4.0, 5.247 mL, 1.433 μmol) was added 30 mM NaOAc pH 4.6 (acceptablerange 4.5-5.0) to give a final protein concentration of 0.88 mg/mL.Intermediate 37 (10 eq., 2.39 mL, 0.014 mmol) was added and the reactionwas mixed at r.t. for 18 hours. Precipitate had formed in the reactionvial. The reaction mixture was split amongst 4×15 mL 10 kDa Amiconcentrifugal filters and each was diluted to 15 mL with 30 mM NaOAc pH4.0. The material was buffer exchanged 4× into 30 mM NaOAc pH 4.0 andsamples were combined to a volume of 25.6 mL, agitating the precipitatein the filter with a pipette tip in between washes. Precipitate remainedin solution so the mixture was let sit at 4° C. overnight. Concentrationwas measured by A280 (30040 cm⁻¹M⁻¹, 27538 g/mol) to be 1.62 mg/mL(100%). UPLC analysis showed 60% recovery of +1FA (Retention time: 4.88min) and +2FA products (Retention time: 5.80 min) (Method J). LCMSmethod T shows desired masses.

Example 19B crude mixture (ratio represented in table below) was testedin vivo and reported in table 1:

% Retention Observed observed time LCMS UPLC (min) UPLC SpeciesCalculated Method T Method J Method J AHA-GDF15 24430 24432 29 3.24AHA-GDF15 + 1 FA 25984 25985 27 4.88 AHA-GDF15 + 2 FA 27538 27540 335.80 AHA-GDF15 + 3 FA 29092 29091 11 6.66

AHA-hGDF15 +1FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on one of the polypeptide chains (on the monomericunit) of the GDF15 homodimer (as represented in embodiment 11B, FormulaH).

AHA-hGDF15 +2FA (Fatty acid) corresponds to a reaction at the N-terminusamino functionality on both polypeptide chains of the GDF15 homodimer(as represented in embodiment 11B, Formula G).

AHA-hGDF15 +3FA (Fatty acid) corresponds to a non-selective reaction atsome other site of the GDF15 homodimer.

Purification:

The crude product was purified by reverse phase chromatography (Buffer A0.1% TFA in water; Buffer B 0.1M TFA in ACN gradient; 99%-80% Buffer A)on a Waters BEH300 130 Å, 3.5 μm, 4.6 mm×100 mm flow rate 2.5 ml/min.

Fraction 1: Unreacted AHA-hGDF15: Rt=17.33 min

Fraction 2: (19B1): AHA-GDF15 +1FA: Rt=20.2 min (approximately 15%yield) (Formula H)

Fraction 3: (19B2): AHA-GDF15 +2FA: Rt=21.6 min (approximately 15%yield) (Formula G)

Fraction 4: (19B3): AHA-GDF15 +3 FA: Rt=23.0 min (approximately 5%yield)

A 1:1 ratio mixture of 19B1 and 19B2 was prepared and tested (19Bm).

Alternatively the reaction may be carried out in 10 mM Na₂HPO₄-7H₂O and30 mM NaOAc at a pH of 4.73: A 10 mg/mL solution of Intermediate 37 inmolecular biology grade water was prepared. To AHA-hGDF15 (Intermediate57, 12.04 mg/mL in 30 mM NaOAc pH 4.0, 4.15 μL, 0.002 μmol) was added 30mM NaOAc 10 mM Na₂HPO₄-7H₂O pH 4.73 to give a final proteinconcentration of 0.88 mg/mL. Intermediate 37 (20 eq., 6.83 μL, 0.041μmol) was added and the reaction was mixed at r.t. for 18 hours. Thereaction mixture had turned cloudy with precipitate. UPLC analysisshowed 58%+1 and +2 products (Method J).

Species Calculated % observed AHA-GDF15 24430 0 AHA-GDF15 + 1 FA 2598411 AHA-GDF15 + 2 FA 27538 47 AHA-GDF15 + 3 FA 29092 34 AHA-GDF15 + 4 FA30646 7

The reaction may also be carried out in 30 mM NaOAc and 10 mM K₂HPO₄ ata pH of 4.6: A 10 mg/mL solution of intermediate 37 in molecular biologygrade water was prepared. To AHA-hGDF15 (intermediate 57, 6.21 mg/mL in30 mM NaOAc pH 4.0, 5.261 mL, 1.337 μmol) was added 30 mM NaOAc 10 mMK₂HPO₄ pH 4.6 (acceptable range 4.5-5.0) to give a final proteinconcentration of 0.88 mg/mL. Intermediate 37 (10 eq., 68.3 μL, 0.409μmol) was added and the reaction was mixed at r.t. for 7 hours. Thereaction mixture had turned cloudy with precipitate. The reactionmixture was split into four 9 mL portions in 15 mL 10 kDa Amiconcentrifugal filter and diluted to 15 mL with 30 mM NaOAc pH 4.0. Thematerial was buffer exchanged 4× into 30 mM NaOAc pH 4.0, agitating theprecipitate between each wash with a pipette tip. The reaction mixturewas concentrated to a volume of 75 mL. Precipitate remained so thematerial was stored at 4° C. for two days. Concentration was measured byA280 (30040 cm⁻¹M⁻¹, 27538 g/mol) to be 0.4 mg/mL (97%). UPLC analysisshowed 61% recovery of +1 and +2 products (Method J).

Observed % observed Species Calculated LCMS method T UPLC Method JAHA-GDF15 24430 24434 34 AHA-GDF15 + 1 FA 25984 25987 34 AHA-GDF15 + 2FA 27538 27540 27 AHA-GDF15 + 3 FA 29092 n/a 5

Reference Example 1: His-hGDF15 BCN (I-58) Conjugated to IntermediatePEG-Myristic Acid Construct Step 1

To a mixture of Azido-PEG23-Amine (30 mg, 0.027 mmol) and myristic NHSester (Toronto Research Chemicals, cat #S69080) (12 mg, 0.037 mmol) wasadded DCM (1 mL) and DIPEA (13 uL), and the mixture was stirred at r.t.overnight. The mixture was purified by silica chromatography elutingwith EtOAc/heptane (0-100%) then MeOH/DCM (0-10%) to give clean productat around 5% MeOH/DCM. LCMS: (Gradient: from 40 to 98% B in 1.4 min−flow1 mL/min Eluent A: water+0.05% formic acid+3.75 mM ammonium acetate,Eluent B: acetonitrile+0.04% formic acid) LCMS: rt=2.20 (Method C) Mass+H calculated: 1354.71 Mass observed: 1354.4.

Step 2

To a solution of BCN-hGDF15 (I-52: 800 uL, 0.25 mg/mL) was added a (2mg/mL in DMSO, 6.3 uL, 10 eq), and the mixture was stirred at r.t.overnight. 1.1 mL 0.20 mg/mL in quantitative yield. (Maldi: +1 masscalculated: 28223 mass observed: 28640; +2 mass calculated: 29543; massobserved: 29962, +3 mass calculated: 30863 mass observed: 31426, +4 masscalculated: 32183 mass observed: 32911).

Reference Example 2: his-hGDF15-PEG23

Degree of Labelling Calculated Observed % His-hGDF15 26468 26360.3 5His-hGDF15-BCN 26644 n/a 0 His-hGDF15 + 1 PEG23 27567 28178.6 15His-hGDF15 + 2 PEG23 28666 29385.1 46 His-hGDF15 + 3 PEG23 29765 30547.228 His-hGDF15 + 4 PEG23 30864 31731.8 5

To a solution of His-hGDF15 BCN (159: 427 μL, 1.17 mg/mL, 0.019 μmol) in30 mM NaOAc pH 4.0 (427 μL) was added azido-dPEG₂₃-amine (QuantaBiodesign, 104 μg, 0.094 μmol). The reaction was mixed at r.t. for 16hours at which point the mixture was exchanged into 30 mM NaOAc pH 4.0using 10 kDa MWCO Amicon centrifugal filter by diluting andconcentrating the sample 5 times to a volume of 140 μL. MALDI analysisshowed full conversion to +1 through +4 products. The concentration wasmeasured by A₂₈₀ (29090 M-1 cm−1, 27600 g/mol) to be 2.099 mg/mL (57%).

Example 20: Apelin Cyclic Peptide BCN Conjugated to Intermediate 47 Step1: Synthesis of pE-R-P-C*-L-S-C*-K-G-P-(D-Nle)-NH(Phenethyl) (DisulfideC⁴-C⁷) (SEQ. ID. NO. 28) Acetate

Preparation of Intermediate 20a

(Loading of 2-Chlorotrityl Chloride Resin with Fmoc-D-Nle-OH, FmocRemoval and Determination of the Loading of the Resin)

2-Chlorotrityl chloride resin (50.0 g, 85.0 mmol) was suspended in ofDCM (400 mL) the suspension was stirred for 10 min and then the solventwas drained, the resin was washed with DCM (3×200 mL). Then a solutionof Fmoc-D-Nle-OH (24.0 g, 68.0 mmol) and DIPEA (96.5 ml, 552.5 mmol) inDCM (120.0 mL) was added to the resin, the suspension was flushed withnitrogen and stirred at rt for 5 min. Another portion of DIPEA (22.7 ml,127.5 mmol) was added and the reaction mixture was stirred at r.t.overnight.

The reaction mixture was drained and the resin was washed with DCM(3×250 mL) for 2 min each time. The resin was quenched with of a mixtureDCM/MeOH/DIPEA (70:15:15) (2×250 mL) for 10 min each time.

The Fmoc group was cleaved by treating the resin with piperidine/DMF(1:3) (1×300 mL) for 5 min. the resin was drained then (1×300 mL) for 15min, followed by washing steps: DMF (6×250 mL, 2 min each time),isopropanol (2×250 mL, 2 min each time) and TBME (6×250 mL, 2 min eachtime). The resin was dried under vacuum at 35° C. for 24 hours to affordIntermediate 20a (57.8 g, loading=1.08 mmol/g).

Preparation of Intermediate 20b

(Assembly of Linear Peptide)

Intermediate 20a (18.5 g, 20.0 mmol) was subjected to solid phasepeptide synthesis on an automatic peptide synthesizer (CSBIO536™). Acoupling cycle was defined as follows:

-   -   Amino acid coupling: AA (3.0 eq.), DIC (3.0 eq.), HOBt (3.0        eq.), DMF (see table below)    -   Washing: DMF (4×150 mL, 2 min each time).    -   Fmoc deprotection: Piperidine/DMF (1:3) (150 mL for 5 min then        150 mL for 15 min).    -   Washing: DMF (6×150 mL, 2 min each time).

Number of couplings × Reaction Coupling Coupling AA time Method 1Fmoc-L-Pro-OH 1 × 120 min DIC/HOBt 2 Fmoc-Gly-OH 1 × 120 min DIC/HOBt 3Fmoc-L-Lys(Boc)-OH 1 × 120 min DIC/HOBt 4 Fmoc-L-Cys(Trt)-OH 1 × 120 minDIC/HOBt 5 Fmoc-L-Ser(tBu)-OH 1 × 120 min DIC/HOBt 6 Fmoc-L-Leu-OH 1 ×120 min DIC/HOBt 7 Fmoc-L-Cys(Trt)-OH 1 × 120 min DIC/HOBt 8Fmoc-L-Pro-OH 1 × 120 min DIC/HOBt 9 Fmoc-L-Arg(Pbf)-OH 1 × 120 minDIC/HOBt 10 Boc-L-Pyr-OH 1 × 120 min DIC/HOBt

After the assembly of the peptide, the resin was washed with DMF (6×150mL, 2 min each time), isopropanol (6×150 mL, 2 min each time) and TBME(6×150 mL, 2 min each time). The peptide resin was dried overnight underhigh vacuum at 35° C. to give Intermediate 20b (57.6 g, 20.0 mmol).

Preparation of Intermediate 20c

(HFIP Cleavage from the Resin)

A portion of Intermediate 20b (27 g, 9.37 mmol) was suspended in DCM(300 mL) and stirred for 15 min. The resin was drained then treated withHFIP/DCM (3:7) (3×270 mL, 15 min each time). The cleavage solution wasfiltered off and collected. The resin was washed with DCM (3×300 mL).The combined cleavage and washing solutions were concentrated to drynessin vacuo. The white powder was dried overnight under vacuum at 35° C.yielding Intermediate 20c-Batch1 (23.5 g, 9.37 mmol).

The above mentioned procedure was repeated with another portion ofIntermediate 20b (28.0 g, 9.72 mmol), affording Intermediate 20c-Batch2(26.1 g, 9.72 mmol).

Preparation of Intermediate 20d

(Solution Phase Coupling of Phenethylamine)

Intermediate 20c-Batch2 (20.0 g, 7.44 mmol, 1.0 eq) and HATU (5.23 g,13.8 mmol, 1.85 eq) were dissolved in DMF (400 mL). A solution ofphenethylamine (1.67 g, 13.8 mmol, 1.85 eq) and DIPEA (3.56 g, 27.6mmol, 3.71 eq) in DMF (60 mL) was added.

The reaction mixture was stirred at rt for 30 min then cooled down to 0°C. then brine (460 mL) was added. The suspension was stirred for 10 minthen the product was isolated by filtration. The filter cake was washedwith H₂O (300 mL), which was then carefully removed, then dissolved inDCM (300 mL). The solution was dried over MgSO4 then concentrated todryness in vacuo. The crude product was subjected to flashchromatography over silica gel (eluents: DCM and DCM/iPrOH (8:2)) toafford Intermediate 20d-Batch1 (14.4 g, 6.6 mmol).

The same procedure was repeated with Intermediate 20c-Batch1 (23.4 g,9.37 mmol), excluding the flash chromatography, affording Intermediate20d-Batch2 (28.0 g, 9.37 mmol).

Preparation of Intermediate 20e

(Protecting Group Removal)

Intermediate 20d-Batch2 (28.0 g, 9.37 mmol) was dissolved inTFA/DCM/EDT/TIS (90:5:2.5:2.5) (290 mL) and the reaction stirred at rtfor 2 h.

The cleavage solution was filtered off and poured onto cold TBME (3 L)(0-4° C.). The turbid suspension was stirred in an ice-water bath for 30min then filtered through a pore 4 glass filter. The white solid thusobtained was washed with TBME (2×100 mL) then dried in vacuum at 35° C.overnight to afford Intermediate 20e-Batch1 (8.9 g, 5.9 mmol).

The same procedure was repeated with Intermediate 20d-Batch1 (14.4 g,6.6 mmol) yielding Intermediate 20e-Batch2 (9.6 g, 6.3 mmol).

Preparation of pE-R-P-C*-L-S-C*-K-G-P-(D-Nle)-NH(Phenethyl) (disulfideC₄-C₇) Acetate

1) Cyclization

Intermediate 20e (5.0 g, 3.3 mmol) was dissolved in water (500 mL). Asolution of iodine (1.18 g, 4.66 mmol, 1.41 eq) in acetic acid (93 mL)was added in one portion. The reaction mixture was stirred at rt for 10min. A solution of ascorbic acid (1.03 g, 5.83 mmol, 1.77 eq) in water(5.8 mL) was added and the reaction mixture stirred for 10 min, filteredand stored at 4° C. until purification.

The same cyclization procedure was repeated until 18.3 g (12.1 mmol) ofIntermediate 20e had been processed.

2) Purification

The solutions of cyclic peptide were subjected to preparative HPLC inportions of 0.5-5.0 g peptide per injection. The fractions having purityhigher than 95% were pooled and freeze dried to yield a total amount of4.89 g (3.2 mmol) of purified peptide (TFA salt) was produced.

3) Acetate Formation by Ion Exchange

75 g (100 mL) of a strong anion exchanger resin (Ion exchanger III,Merck) in its OH⁻ form was placed in sintered glass filter (porosity 3)and then a solution of acetic acid/water (1:3) (300 mL) was added, thesuspension was manually stirred for 2 min then the resin was drained.The process was repeated with another portion of acetic acid/water (1:3)(300 mL). The resin was washed with deionized water until a neutraldrain was observed. Then the resin was transferred to a 4×20 cm columnequipped with a sintered glass filter (porosity 3).

4.8 g of purified peptide was dissolved in deionized water (50 mL) andadded to the column. The product was eluted with deionized water (200mL). Control of product elution was done by TLC spotting, the richfractions were pooled and freeze dried to givepE-R-P-C*-L-S-C*-K-G-P-(D-Nle)-NH(Phenethyl) (disulfide C⁴-C⁷) (SEQ. ID.NO: 28) Acetate (4.1 g, 2.9 mmol).

The pure product was analyzed by analytical HPLC (Analytical method F;t_(R)=8.01 min) and UPLC-HRMS (Analytical method G; measured:[M+2H]²⁺=643.328; calculated: [M+2H]²⁺=643.324). The acetate content was7.99-8.27% and the water content was 1.94-1.96%.

Step 2: Preparation ofpE-R-P-C*-L-S-CP-N⁶-[[(1α,8α,9α)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl]-K-G-P-(D-Nle)-NH(Phenethyl)[disulfide C⁴-C⁷] (SEQ. ID. NO: 30)

A mixture of pE-R-P-C*-L-S-C*-K-G-P-(D-Nle)-NH(Phenethyl) triacetate[disulfide C⁴-C⁷] (100 mg, 0.068 mmol), sodium bicarbonate (38 mg, 0.452mmol) and water (83 uL) in DMF (1 mL) was stirred at RT for 10 mins,then (1R,8S)-bicyclo[6.1.0]non-4-yn-9-ylmethyl succinimidyl carbonate(Berry &associates, 20 mg, 0.068 mmol) was added. The reaction mixturewas stirred at RT for 90 mins. 1 mL of water was added to the mixture,and the resultant solution was lyophilized to give a powder which wasused for the next step without further purification.

Step 3: Preparation of Example 20

A mixture ofpE-R-P-C*-L-S-C*-N⁶-[[(1α,8α,9α)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl]-K-G-P-(D-Nle)-NH(Phenethyl)[disulfide C⁴-C⁷] (SEQ. ID. NO: 30) (50 mg of the product from Step 2 in1 mL of water, 0.034 mmol) and Intermediate 47 (52 mg, in 268 uL ofwater) was stirred at RT for about 3 hrs. The reaction mixture was thenpurified by preparative HPLC (Sunfire 30×50 mm 5 um column ACN/H₂Ow/0.1% TFA 75 ml/min, 15-40% ACN 5 min gradient). The product fractionwas lyophilized to give the titled product as TFA salt (24 mg, 21%).LCMS (Waters Acquity UPLC BEH C18 1.7 um 2.1×50 mm, 50° C., Eluent A:Water+0.1% Formic Acid, Eluent B: Acetonitrile+0.1% Formic Acid,gradient 2% to 98% B/A over 5.15 mins): Rentention time: 2.77 mins; MS[M+2]²⁺: observed: 1491.8808, calculated: 1491.8560.

Example 21A: Apelin Cyclic Peptide BCN Conjugated to Intermediate 47Step 1

A mixture of pE-R-P-R-L-C*-H-K-G-P-Ne-C*-F-OH(Disulfide C⁶-C¹²) (SEQ. D.NO: 29) 50 g, 0.033 mmol, as prepared in U.S. Pat. No. 8,673,848),sodium bicarbonate (18 mg, 0.215 mmol) and water (40 uL) in DMF (0.5 mL)was stirred at RT for 10 mins, then(1R,8S)-bicyclo[6.1.0]non-4-yn-9-ylmethyl succinimidyl carbonate (Berry&associates, 18 mg, 0.065 mmol) was added. The reaction mixture wasstirred at RT for 90 mins. A mixture of +1 and +2 additions was observedby LCMS, so mixture was purified by mass triggered HPLC (Peptide Method5 25-50% ACN 5 min gradient: Conditions: Sunfire 30×50 mm 5 um columnACN/H2O w/0.1% TFA 75 ml/min 1.5 ml injection): rt 3.2 min (+1), rt 4.65min, 4.9 min (+1 and +2 mixture). LCMS confirms desired +1 product in61% yield and +1, +2 mixture in 18% yield. LCMS: (Basic Eluent A:Water+5 mM Ammonium Hydroxide Eluent B: ACN Acidic Column: Sunfire C183.5 μm 3.0×30 mm−40° C. Basic Column: XBridge C18 3.5 μm 3.0×30 mm−40°C.) Retention time: 0.98 mins; MS [M+2]²⁺: observed: 856.0, calculated:865.0245.

Step 2

A mixture ofpE-R-P-R-L-C*-H-N⁶-[[(1α,8α,9α)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl]-K-G-P-Nle-C*-F-OH(DisulfideC⁶-C¹²) (SEQ. ID. NO: 31) (21.33 mg, 0.014 mmol) and intermediate 47 (24mg, 0.014 mmol) was stirred at RT for about 3 hrs. The reaction wascomplete by LCMS and was lyophilized to give the titled product (23 mg,48%). LCMS (Waters Acquity UPLC BEH C18 1.7 um 2.1×50 mm, 50° C., EluentA: Water+0.1% Formic Acid, Eluent B: Acetonitrile+0.1% Formic Acid,gradient 2% to 98% B/A over 5.15 mins): Rentention time: 2.22 mins; MS[M+2]²⁺: observed: 1616.9464, calculated: 1616.976.

Example 21B: Apelin Cyclic Peptide Conjugated to Fatty Acid-LinkerConstruct I-37 Step 1: Synthesis ofA-H-Q-R-P-C-L-S-C-K-G-P-Dnle-Phenethyl amine Intermediate 21B1 (SEQ. ID.NO: 32)

Phenethylamine-AMEBA resin (Sigma Aldrich, 0.1 mmol, 1.0 mmol/g) wassubjected to solid phase peptide synthesis on an automatic peptidesynthesizer (CEM Liberty Blue Microwave) with standard double Arg forthe Arg residues and Dnle coupled double time. Amino acids were preparedas 0.2 M solutions in DMF. A standard coupling cycle was defined asfollows:

-   -   Amino acid coupling: AA (5 eq.), HATU (5 eq.), DIEA (25 eq.)    -   Washing: DMF (3×7 mL)    -   Fmoc Deprotection: 20% Piperidine/0.1 M HOBt (2×7 mL)    -   Washing: DMF (4×7 mL then 1×5 mL)

Number of couplings × Reaction time Coupling Coupling AA (Temp) Method 1Fmoc-D-Nle- 1 × 10 min (70° C.)  DIEA/HATU OH 2 Fmoc-L-Pro- 1 × 5 min(70° C.) DIEA/HATU OH 3 Fmoc-L-Gly- 1 × 5 min (70° C.) DIEA/HATU OH 4Fmoc-L-Lys- 1 × 5 min (70° C.) DIEA/HATU OH 5 Fmoc-L-Cys- 1 × 5 min (70°C.) DIEA/HATU OH 6 Fmoc-L-Ser- 1 × 5 min (70° C.) DIEA/HATU OH 7Fmoc-L-Leu- 1 × 5 min (70° C.) DIEA/HATU OH 8 Fmoc-L-Cys- 1 × 5 min (70°C.) DIEA/HATU OH 9 Fmoc-L-Pro- 1 × 5 min (70° C.) DIEA/HATU OH 10Fmoc-L-Arg- 2 × 25 min (25° C.)  DIEA/HATU OH 11 Fmoc-L-Gln- 1 × 5 min(70° C.) DIEA/HATU OH 12 Fmoc-L-His- 1 × 5 min (70° C.) DIEA/HATU OH 13Fmoc-L-Ala- 1 × 5 min (70° C.) DIEA/HATU OH

After the assembly of the peptide, the resin was washed with DMF (2×50mL) and DCM (2×50 mL) then dried under vacuum to give Intermediate 21B1(276 mg, 0.1 mmol).

Step 2: Preparation of Intermediate 21B2 (Cleavage of Peptide fromResin)

Intermediate 21B1 (276 mg, 0.1 mmol) was combined with 4 mL TFA solution(37 mL TFA, 1 mL H₂O, 1 mL TIPS, 3.06 g DTT) and shaken at r.t. for 3hours. The solution was removed from the resin and precipitated into 40mL cold Et₂O. The solution was vortexed and let stand over ice for 10minutes before centrifuging at 4000 rpm for 5 minutes. The solvent wasremoved and the white solid was washed twice more with cold Et₂O (40 mLeach time), centrifuged (5 minutes each time) and decanted. The solidwas dried under vacuum overnight yielding Intermediate 21B2 (17.4 mg,0.012 mmol). LCMS (SQ2 ProductAnalysis-Acidic-Peptide-Polar, AcquityUPLC BEH C18 column, 130 Å, 1.7 μm, 2.1 mm×50 mm, 50° C.): R_(t)=1.83minutes, MS [M+H]1513.5.

Step 3: Preparation of Intermediate 21B3 (Cyclization of CysteineResidues)

Intermediate 21B1 (29.6 mg, 0.020 mmol) was dissolved in water (3 mL)and 10 drops of DMSO to give a slightly cloudy solution. Iodine (50 mMin HOAc, 0.783 mL, 0.039 mmol) was added slowly dropwise and thereaction was mixed at r.t. overnight. LCMS analysis of the crudereaction showed complete conversion of starting material. 0.5 M ascorbicacid was added dropwise until color dissipated. The material waspurified via MS-triggered HPLC. Lyophilization of the pooled fractionsgave 7 mg of the desired product as a white powder (4.63 μmol, 24%).LCMS (SQ2 ProductAnalysis-Acidic-Peptide, Acquity UPLC BEH C18 column,130 Å, 1.7 μm, 2.1 mm×50 mm, 50° C.): R_(t)=0.90 minutes, MS [M+H]1511.8.

Step 4: Preparation of Conjugate Comprising ApelinA-H-Q-R-P-C-L-S-C-K-G-P-Dnle-Phenethyl Amine (SEQ. ID. NO. 321 andIntermediate I-37 (N-terminus conjugation)—Example 21B

A 10 mg/mL solution of NHS-fatty acid was prepared in H₂O. Intermediate21B3 (1.5 mg, 0.993 μmol) was dissolved in 30 mM pH4 NaOAc buffer (672μL) and NHS-fatty acid (I-37: 0.850 mL, 5.10 μmol) was added. Thereaction was mixed at r.t. for 16 hours at which point an additional 1.5mg of NHS-fatty acid (10 mg/mL in H₂O) was added and the reaction mixedat r.t. for 16 hours. 8 mg of NHS-fatty acid (10 mg/mL in H₂O) was addedand the reaction mixed at r.t. for 3 days and 1.7 mg of NHS-fatty acid(10 mg/mL in H₂O) was added. The mixture was shaken at r.t. for 16 hoursand purified via M-triggered HPLC to give 1.7 mg of the title compoundas a white powder (0.510 μmol, 51%). LCMS (SQ2ProductAnalysis-Acidic-Peptide-Polar, Acquity UPLC BEH C18 column, 130Å, 1.7 μm, 2.1 mm×50 mm, 50° C.): R_(t)=3.87 minutes, MS [M+H+2/2]1533.1; [M+H+3/3] 1022.9.

Examples 22 to 24 Refers to Conjugates of a Fatty Acid with a siRNA

Kits for siRNA synthesis are commercially available, e.g., from NewEngland Biolabs and Ambion. A siRNA agent can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, siRNA agent can be chemically synthesizedusing naturally-occurring nucleotides or variously modified nucleotidesdesigned to decrease off-target effects, and/or increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. “G,” “C,” “A,” “T” and “U” each generally stand for a nucleotidethat contains guanine, cytosine, adenine, thymidine and uracil as abase, respectively. However, the terms “ribonucleotide”,“deoxynucleotide”, or “nucleotide” can also refer to a modifiednucleotide or a surrogate replacement moiety.

Those skilled in the art will appreciate that it is possible tosynthesize and modify the siRNA as desired, using any conventionalmethod known in the art (see Henschel et al. 2004 DEQOR: a web-basedtool for the design and quality control of siRNAs. Nucleic AcidsResearch 32 (Web Server Issue): W113-W120).

Example 22: Conjugation of a Fatty Acid Moiety of Formula A3 to siRNA

Preparation of Intermediate 22a:

From TTR siRNA [siRNA to transthyretin (TTR), synthesized usingconventional methods known in the art] was converted to 22a usingstandard oligonucleotide procedures (e.g. reaction with6-(4-Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite(Glen Research Catalog No: 10-1906))

TTR siRNA:

Antisense strand: Puagagcaagaacacugu*u*rX058 (SEQ. ID. NO: 18) wherein:

P is phosphate, lowercase letters indicate a 2′-OMe modified nucleotide,underlined letters indicate a 2′-F modified nucleotide, italics lettersindicate a 2′-MOE modified nucleotide, r is a basic ribitol, * refers toa phosphorothioate linkage, and X058 is a non-nucleotidic 3′ end cap ofFormula:

Sense Strand: aacaguguucuugcucuar-C60H (SEQ. ID. NO: 19).

-   -   refers to a phosphate; and C6OH (also known as C6) is a        non-nucleotidic 3′ end cap of Formula:

Preparation of Intermediate 22b:

To a 0.556 ml of a solution of siRNA 22a (TTR siRNA 14.02 mM in H2O,7.79 μmol) at 0° C., 0.556 ml DMF was added and warmed up to roomtemperature. Then 208 μl TEA (0.3M in DMF, 62 μmol) was added and 260 μlBCN-NHS ((1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidylcarbonate) (0.15M in DMF, 39 μmol). The resulted reaction stirred atroom temperature for 1 hr. Analytical HPLC showed the disappearance ofstarting material 22a. The reaction was diluted to 10 ml with de-ionizedH₂O and became cloudy. The above mixture was extracted with ethylacetate5 times to remove small organic molecules. The aqueous layer wasseparated and lyophilized. The dried product solid (22b) was used as itwas.

Preparation of Example 22

Mixed 80 μl Compound 22b (12.3 mM in H₂O, 0.968 μmol) and 65 μl bisfatty acid-N₃ (22c: step 2 of Example 15: 44.7 mM in DMSO, 2.90 μmol).The resulted mixture was cloudy, so 80 μl 1:1 DMF/THF was added and thereaction was still slightly cloudy. Another 80l DMSO was added to get aclear solution. The reaction stirred at room temperature for overnight.The reaction was diluted with de-ionized H2O and purified over HPLC toafford 5.6 mg compound 22 (65.9% yield). HPLC conditions forpurification: Column: Xselect Prep phenylhexyl 5 um OBD 19×50 mm;organic solvent: ACN modified with 100 mM TEA.HOAc; aqueous solvent: H₂Omodified with 100 mM TEA.HOAc; Gradient: 5-60% AcCN/H₂O; Time: 10 min.Under LC-MS method H, the product showed a single peak with retentiontime of 5.44 min with the desired MW of 8778 after deconvolusion.

Example 23: Conjugation of a Fatty Acid Moiety of Formula A1 to siRNAPreparation of Compound 4

Example 23 was prepared using the same procedure as example 22.

Preparation of 23a:

To a solution NHS-fatty acid (20 mg) in DCM (16 mL) was addedazido-peg7-amine (QuantaBiodesign, cat #10523) (31 mg) and DIPEA (52 uL)and the mixture was stirred at r.t. for 2 h. Complete conversion wasobserved by LC-MS analysis. The mixture was concentrated, re-dissolvedin MeOH (3 mL) and purified by MS triggered HPLC with 0.1% TFA (rt=1.59min, mass (M+1) expected: 818.062 mass observed: 817.9) to give cleanproduct. Half material was lost due to 800 Da cut-off set in the HPLC MSsystem. 5-10 mg (17-33%) clean product was obtained.

Mixed 80 μl 22b (12.3 mM in H₂O, 0.968 μmol) and 651 tris fatty acid-N3(23a: 44.7 mM in DMSO, 2.90 μmol). The reaction afforded 5.2 mg compound23 (58.0% yield). Under LC-MS method H, the product showed a single peakwith retention time of 5.87 min with the desired MW of 9257 afterdeconvolusion.

Example 24: Conjugation of Fatty Acid of Formula A3 to APOCIII siRNA

Preparation of 24b

To a solution of 24a (1.244 g, 4.19 mmol) in 25 ml DCM, TEA (0.58 ml,4.19 mmol) was added, followed by N3-pentanoic acid (500 mg, 3.49 mmol).EDC (804 mg, 4.19 mmol) was added at last. The reaction stirred at roomtemperature for 4 hr. The reaction was extracted between brine and DCM.Combined all organics, dried, concentrated and purified over SiO₂ gelwith 60% ethylacetate/heptane to afford 1.20 g compound 24b (89% yield).¹H NMR (CHLOROFORM-d, 400 MHz) d: 5.88-6.37 (m, 1H), 4.60 (d, J=4.8 Hz,2H), 3.77 (s, 3H), 3.33 (t, J=6.7 Hz, 2H), 3.13 (d, J=6.5 Hz, 2H), 2.30(t, J=7.2 Hz, 2H), 1.81-1.95 (m, 1H), 1.63-1.81 (m, 5H), 1.48-1.57 (m,2H), 1.46 (s, 9H), 1.36 (d, J=6.8 Hz, 2H)

Preparation of 24c

To a solution of 24b (860 mg, 2.23 mmol) in 12 ml THF, 1N NaOH (5.58 ml,5.58 mmol) was added. The reaction stirred at room temperature for 0.5hr. The reaction was diluted with brine and 20 ml DCM was added. The pHof aqueous layer was adjusted to pH ˜5 with 1N HCl, then extracted withDCM. Combined all organics, dried, concentrated and and the crude solidwas redissolved into 6 ml DCM. 0.6 ml TFA was added and the reactionstirred at room temperature for 2 hr. The reaction was concentrated andafforded the crude 24c (400 mg, 54%), which was used directly withoutfurther purification. Under LC-MS method I, the product showed a majorpeak at 0.43 min. with a mass of 272.5 (M+H⁺).

Preparation of 24e

To a solution of 24c (400 mg, 1.04 mmol) in 10 ml DCM, TEA (0.434 ml,3.11 mmol) was added, followed by the addition of 24d (538 mg, 1.35mmol). The reaction stirred at room temperature for 3 hr. The reactionwas extracted between H2O and DCM. Combined all organics, dried,concentrated and purified over SiO2 gel with 8% MeOH/DCM to afford 475mg of 24e (82% yield). ¹H NMR (CHLOROFORM-d, 400 MHz) d: 6.74-6.98 (m,1H), 5.95-6.13 (m, 1H), 4.55 (dd, J=7.2, 2.4 Hz, 2H), 3.32 (t, J=6.7 Hz,4H), 3.11 (d, J=5.8 Hz, 2H), 2.30-2.37 (m, 2H), 2.20-2.26 (m, 2H), 1.90(br. s., 2H), 1.71-1.80 (m, 2H), 1.59-1.71 (m, 4H), 1.51-1.59 (m, 2H),1.35-1.51 (m, 13H), 1.20-1.35 (m, 13H)

Preparation of 24f

Preparation of Intermediate 24f2

To a solution of 24f1 (1.0 g, 4.97 mmol) in MeOH (40 ml), TEA (1.04 ml,7.45 mmol) was added, followed by di t-butyl dicarbonate (2.17 g, 9.94mmol). The reaction was heated at 60° C. for 1.5 hr. The reaction wasconcentrated and purified over SiO₂ column with 5% MeOH/DCM to afford1.2 g compound 24f2 (80% yield). ¹H NMR (CHLOROFORM-d, 400 MHz) d: 4.51(br. s, 1H), 3.00-3.22 (m, 2H), 2.37 (t, J=7.4 Hz, 2H), 1.59-1.71 (m,2H), 1.46 (s, 11H), 1.29 (br. s., 12H).

Preparation of Intermediate 24f3

To a solution of 24f2 (1.2 g, 3.98 mmol) in DCM (30 ml), N-hydroxylsuccinimide (0.60 g, 5.18 mmol) was added, followed by EDC (1.0 g, 5.18mmol). The solution was stirred at room temperature for overnight. Thereaction was concentrated, directly loaded onto SiO2 column and purifiedwith 40% ethylacetate/heptane to afford 1.46 g compound 24f3 (92%yield). ¹H NMR (CHLOROFORM-d, 400 MHz) d: 4.49 (br. s, 1H), 3.04-3.19(m, 2H), 2.86 (d, J=4.5 Hz, 4H), 2.62 (t, J=7.4 Hz, 2H), 1.70-1.82 (m,2H), 1.37-1.53 (m, 13H), 1.30 (br. s., 10H)

Preparation of Intermediate 24f

To a solution of GalNAc3-NH2 (300 mg, 0.16 mmol) in DCM (1.5 ml), TEA(0.11 ml, 0.79 mmol) was added, followed by the addition of 24f3 (188mg, 0.47 mmol). The reaction stirred at room temperature for overnight.Then trifluoroacetic acid (1.0 ml) was added. After 2 hrs, LC-MS showedthe disappearance of the intermediate. The reaction was concentrated andpurified on open access HPLC under acidic condition with ELSD as adetection. The HPLC fractions containing the product were collected andthe solvent was evaporated to afford 220 mg compound 24f (67% yield).LC-MS showed that partial product lost one acetyl group. HPLC conditionsfor purification: column: Sunfire 30×100 mm 5 um column; organicsolvent: ACN w/7.5% TFA; aqueous solvent: H₂O w/7.5% TFA; flow rate: 75ml/min. Gradient: 15-40% H₂O/AcCN; Time: 9.5 min. detection: ELSD(Evaporative Light Scattering Detector) as detection. Under LC-MS methodI, the product showed a peak at 0.83 min. with a mass of 989.9 (M/2+H⁺).

Preparation of 24 g

To a solution of 24e (172 mg, 0.31 mmol) in 3 ml DCM, 24f (250 mg, 0.12mmol) was added, followed by TEA (0.069 ml, 0.50 mmol). EDC (95 mg, 0.5mmol) was added at last. The reaction stirred at room temperature forovernight. The reaction was concentrated and purified over SiO₂ columnwith 5% MeOH/DCM to afford 230 mg 24 g (74% yield). Under LC-MS methodI, the product showed a peak 1.30 min. with a mass of 1258.2 (M/2+H+).

Preparation of 24 h

To a solution of 24 g (230 mg, 0.091 mmol) in 1 ml THF, 0.457 ml 4N HCl(in dioxane, 1.83 mmol) was added. The reaction stirred at roomtemperature for 1 hr. The reaction was concentrated and purified overHPLC to afford 50 mg 24 h (23% yield), which lost one acetyl group onthe sugar. HPLC conditions for purification: column: Sunfire 30×100 mm 5um column; organic solvent: ACN w/7.5% TFA; aqueous solvent: H₂O w/7.5%TFA; flow rate: 75 ml/min. Gradient: 15-40% H2O/AcCN; Time: 9.5 min.detection: ELSD (Evaporative Light Scattering Detector) as detection.Under LC-MS method I, the product showed a peak at 0.95 min. with a massof 1187.1 (M/2+H+).

Preparation of 24i

To a solution of 2,2,13,13-tetramethyltetradecanedioic acid (40 mg,0.127 mmol) in 2 ml DCM, N—OH succinimide (9.81 mg, 0.085 mmol) wasadded, followed by the addition of EDC (16.34 mg, 0.085 mmol). Thereaction was stirred at room temperature for overnight. The reaction wasconcentrated and purified over HPLC to afford 20 mg 24i (38% yield).HPLC conditions for purification: column: Sunfire 30×100 mm 5 um column;organic solvent: ACN w/7.5% TFA; aqueous solvent: H₂O w/7.5% TFA; flowrate: 75 ml/min. Gradient: 45-70% H2O/AcCN; Time: 9.5 min. detection:ELSD (Evaporative Light Scattering Detector) as detection. Under LC-MSmethod I, the product showed a peak at 1.55 min. with a mass of 434.3(M+Na⁺).

Preparation of 24j

To a solution of 24 h (20 mg, 8.05 μmol) in 0.5 ml DCM, TEA (4.5 μl, 32μmol) was added, followed by the addition of 24i (6.62 mg, 16 μmol) andDMAP (3.93 mg, 32 μmol). The reaction stirred at room temperature forovernight. Then 0.5 ml 2N MeNH₂/MeOH was added for the deprotection. Thereaction stirred at room temperature for another overnight. The reactionwas concentrated and acetone was added to precipitate the product andremove excess reagents and lipids to afforded 12 mg 24j (64% yield).Under LC-MS method I, the product showed a peak at 0.69 min. with a massof 1166.9 (M/2+H⁺).

Preparation of 24K

Step 1

APOCIII siRNA [siRNA to gene APOCIII (also known as APOC3 or Apoc 3),synthesized using conventional methods known in the art]:

Annealing Oligonucleotides

General Procedure:

Each oligonucleotide pellet is briefly spinned down in a centrifuge anddissolved in Duplex Buffer (100 mM Potassium Acetate; 30 mM HEPES, pH7.5) at high concentration (1-10 OD260 per 100 mL buffer). Heating (upto 94° C.) and vortexing may be used to facilitate resuspension. Thesense and the antisense strands are then added together in equimolaramounts. The mixed oligonucleotides are then heated to 94° C. andgradually cooled down. For sequences with significant secondarystructure, a more gradual cooling/annealing step may be employed. Thisis easily done by placing the oligo solution in a water bath or heatblock and unplugging/turning off the machine. The resulting product willbe in a stable, double-stranded form and can be stored at 4° C. orfrozen.

Antisense strand of APOCIII siRNA comprises a sequence of APOCIII,wherein the 3′ end of the strand terminates in a phosphate and furthercomprises, in 5′ to 3′ order, a ribitol, another phosphate, and a 3′ endcap X058, a non-nucleotidic 3′ end cap of Formula:

The sense strand comprises a sequence of APOCIII complementary to theantisense strand, wherein the 3′ end of the strand terminates in aphosphate and further comprises, in 5′ to 3′ order, a ribitol, anotherphosphate, and a 3′ end cap C6OH, a non-nucleotidic 3′ end cap ofFormula:

APOCIII siRNA was reacted with with2-Dimethoxytrityloxymethyl-6-fluorenylmethoxycarbonylamino-hexane-1-succinoyl-longchain alkylamino-CPG (Glen Research Catalog No 20-2957) to generateproduct 24 k1.

Step 2

APOCIII siRNA (24 k1: 401 μL, 11.2 mM in H₂O, 4.49 μmol) was mixed with401 μl DMF to get a clear solution. Then TEA (180 μl, 0.25M in DMF, 45μmol) was added, followed by BCN-NHS((1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate)(9.16 mg, 31 μmol). The reaction stirred at room temperature for 1 hr.The reaction was diluted with H2O to 10 ml and extracted withethylacetate 3 times. The aqueous layer was separated and concentratedto ˜5 ml and purified over PD-10 desalting column (GE healthcare).

Preparation of 24

24j (5.8 mg, 2.5 μmol) was added into 105 μl compound 24k (Apoc 3 siRNA9.5 mM in H₂O, 0.993 μmol). After 1 hr, the reaction became viscous. Soanother 60 μl H₂O was added and the reaction stirred at room temperaturefor overnight. The reaction was diluted with H₂O and purified over HPLCto afford 5 mg conjugate 24 (57% yield). HPLC conditions forpurification: Column: Xselect Prep phenylhexyl 5 um OBD 19×50 mm;organic solvent: AcCN modified with 100 mM TEA.HOAc; aqueous solvent:H₂O modified with 100 mM TEA.HOAc; Gradient: 5-50% AcCN/H2O; Time: 10min. Under LC-MS method H, the product showed a peak at 5.96 min. withthe desired mass of 8896 after deconvolution.

Reference Example 3: siRNA Conjugated with GalNAc

Reference Example 3 was prepared according to procedure of Example 24(replacing 24j with GalNac3-N3 (below).

Preparation of GalNac3-N3

Preparation of Intermediate 2

Compound 1 (2.06 g, 1.07 mmol) was dissolved in 20 ml ethanol, TFA (82ul, 1.07 mmol) was added, followed by 10% Pd/C (0.114 g, 0.1 mmol). Thereaction was treated under H₂ balloon for 6 hours. The reaction wasfiltered, washed with ethanol and concentrated to get white solid, whichwas used directly for next step. Under LC-MS method I, the productshowed a peak at 0.81 min. with a mass of 898.5 (M/2+H+).

Preparation of Intermediate 3

Compound 2 (4.848 g, 0.479 mmol) was dissolved in 10 ml anhdrous DMF,2,5-dioxopyrrolidin-1-yl-4 azidobutanoate (0.325 g, 1.436 mmol) wasadded, followed by the addition of DIPEA (0.418 ml, 2.394 mmol). Thereaction was to react overnight at room temperature. The reaction wasconcentrated with no heating, directly loaded onto a pre-equilibratedSiO2 column and purified with 0-20% methanol/DCM step gradient to afford2.383 g compound 3 (49.25% yield). Under LC-MS method I, the productshowed a peak at 1.27 min. with a mass of 953.7 (M/2+H⁺).

Preparation of Intermediate 4

Mixed compound 3 (1.33 g, 0.70 mmol) with MeNH2 (17.45 ml, 2.0M inMethanol, 34.9 mmol). The reaction stirred at room temperature for 2 hr.LC-MS only showed the product peak. The reaction was concentrated. Thenthe solid redissolved into ethanol and was precipitated with acetone toafford 1.0 g compound 4 (94% yield). Under LC-MS method I, the productshowed a peak at 0.53 min. with a mass of 764.5 (M/2+H+).

Example 25: Conjugation of Carrier Protein (CRM197) and a Fatty AcidStep 1:2-((2,2-dimethyl-4-oxo-3,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-5-azatetracontan-40-yl)carbamoyl)-2-undecyltridecanedioicAcid (25a)

t-boc-N-amido-dPEG®₁₁-amine (100 mg, 0.155 mmol, Quanta Biodesign) andIntermediate 5 (80 mg, 0.148 mmol) were dissolved in THF (3 mL) andstirred at room temperature under nitrogen nitrogen. After 30 minutes,DIPEA (0.05 mL, 0.286 mmol) was added and the reaction mixture stirredat room temperature overnight. Complete conversion was observed by LCMS(Acidic Eluent A: Water+0.05% Trifluoroacetic Acid, Eluent B: ACN,column Sunfire C18 3.5 μm 3.0×30 mm−40° C., 5-95% gradient 2 minutes,retention time 1.92 min). The reaction mixture was concentrated underreduced pressure, then dissolved in about 1.5 mL of acetonitrile.Purified on a MS-triggered HPLC (Sunfire 30×50 mm 5 um column ACN/H₂Ow/0.1% TFA 75 ml/min 1.5 ml injection, 65-95% ACN 3.5 min gradient,retention time 3.23 minutes) and the fractions pooled and lyophilized togive 85 mg clean product in 54% yield. Clear oil. LCMS: Method D Rt=1.18min, M+H 1070.1; ¹H NMR (400 MHz, ACETONITRILE-d₃) δ ppm 0.82-1.03 (m,1H) 1.11-1.37 (m, 10H) 1.37-1.51 (m, 2H) 1.51-1.64 (m, 1H) 1.69-1.82 (m,1H) 1.90-2.04 (m, 66H) 2.05-2.21 (m, 8H) 2.21-2.42 (m, 1H) 3.17-3.28 (m,1H) 3.40-3.68 (m, 13H).

Step 2:2-((35-amino-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)carbamoyl)-2-undecyltridecanedioicAcid (25b)

25a (5 mg, 4.68 μmol) was dissolved in DCM (Volume: 2 mL), thentrifluoroacetic acid (25 μl, 0.324 mmol) was added. The reaction mixturewas stirred at room temperature under nitrogen atmosphere for about 2hours. Complete conversion was observed by LCMS (Acidic Eluent A:Water+0.05% Trifluoroacetic Acid, Eluent B: ACN, column Sunfire C18 3.5μm 3.0×30 mm −40° C., 5-95% gradient 2 minutes, retention time 1.45min). The reaction mixture was concentrated under reduced pressure, thenrinsed with DCM and concentrated again 3 times. Dissolved in a mixtureof acetonitrile and DMSO. Purified on a MS-triggered HPLC (Sunfire 30×50mm 5 um column ACN/H2O w/0.1% TFA 75 ml/min 1.5 ml injection, 45-70% ACN3.5 min gradient, retention time 2.50 minutes) and the fractions pooledand lyophilized to give 2.5 mg clean product in 55% yield. Clear oil.

Method A Rt=1.45 min, M+H 969.9; ¹H NMR (400 MHz, ACETONITRILE-d₃) δ ppm0.62-0.91 (m, 2H) 0.91-1.10 (m, 3H) 1.10-1.31 (m, 18H) 1.46 (quin,J=7.21 Hz, 2H) 1.59-1.89 (m, 35H) 1.94-2.09 (m, 1H) 2.16 (t, J=7.40 Hz,2H) 2.97-3.11 (m, 1H) 3.24-3.37 (m, 1H) 3.37-3.61 (m, 28H) 3.61-3.89 (m,2H) 7.85 (br. s., 1H).

Step 3:2-(((S)-5-(3-amino-3-oxopropyl)-3,6-dioxo-1-phenyl-2,10,13,16,19,22,25,28,31,34,37,40-dodecaoxa-4,7-diazadotetracontan-42-yl)carbamoyl)-2-undecyltridecanedioicAcid (25d)

A solution of 25b (20 mg, 0.018 mmol) in THF (Volume: 2 mL) was added toZ-L-Gln-Osu 25c (Santa Cruz Biotechnology, CAS 34078-85-8, 11 mg, 0.029mmol), then DIPEA (75 μl, 0.429 mmol) was added. Stirred at roomtemperature under a nitrogen atmosphere over weekend. Completeconversion was observed by LCMS (Acidic Eluent A: Water+0.05%Trifluoroacetic Acid, Eluent B: ACN, column Sunfire C18 3.5 μm 3.0×30mm−40° C., 5-95% gradient 2 minutes, retention time 1.77 min). Thereaction mixture was concentrated under reduced pressure, then dissolvedin acetonitrile. Purified on a MS-triggered HPLC (Sunfire 30×50 mm 5 umcolumn ACN/H₂O w/0.1% TFA 75 ml/min 1.5 ml injection, 55-80% ACN 3.5 mingradient, retention time 2.70 minutes) and the fractions pooled andlyophilized to give 10.5 mg clean product in 46% yield as a clearcolorless oil. Method C Rt=1.60 min, M+H 1232.4; ¹H NMR (400 MHz,ACETONITRILE-d₃) b ppm 0.67-0.93 (m, 2H) 0.93-1.10 (m, 2H) 1.10-1.32 (m,15H) 1.45 (quin, J=7.24 Hz, 1H) 1.59-1.69 (m, 1H) 1.75-1.93 (m, 30H)1.94-2.21 (m, 20H) 3.23 (quin, J=5.26 Hz, 1H) 3.28-3.51 (m, 23H) 3.95(td, J=7.73, 5.44 Hz, 1H) 4.92-5.22 (m, 1H) 5.78 (br. s., 1H) 6.13-6.42(m, 1H) 6.88 (br. s., 1H) 7.20-7.36 (m, 2H) 7.42 (t, J=5.07 Hz, 1H).

Step 4: mTGase-Mediated Labelling of CRM197 with Fatty Acid (Example 25)

To a solution of 25d in 100 mM tris buffer pH 8 (8 mg/mL, 203 μL, 1.316μmol) was added CRM197 (33 mg/mL, 1.515 μL, 0.00086 μmol) followed by asolution of transglutaminase enzyme (Ajinomoto) in PBS (50 mg/mL, 0.455μL, 0.00060 μmol). The reaction was stirred at r.t. for 16 hours. Thereaction mixture was exchanged into 100 mM tris buffer pH 8 using 10 kDaMWCO Amicon centrifugal filter by diluting and concentrating thereaction 5 times to a volume of 100 μL. LCMS analysis showed conversionto +1, +2, +3 and +4 products. LCMS QT2; Protein_35-70 kDa_3 min:R_(t)=1.45 min; MS [M+25d]: observed: 59625, calculated: 59624; MS[M+(2×25d)]: observed: 60839, calculated: 60838; MS [M+(3×25d)]:observed: 62054, calculated: 62052; MS [M+(4×25d)]: observed: 63270,calculated: 63266.

CRM197 Sequence:

(SEQ. ID. NO: 20) GADDVVDSSK SFVMENFSSY HGTKPGYVDS IQKGIQKPKS GTQGNYDDDWKEFYSTDNKY DAAGYSVDNE NPLSGKAGGV VKVTYPGLTK VLALKVDNAE TIKKELGLSLTEPLMEQVGT EEFIKRFGDG ASRVVLSLPF AEGSSSVEYI NNWEQAKALSVELEINFETR GKRGQDAMYE YMAQACAGNR VRRSVGSSLS CINLDWDVIR DKTKTKIESLKEHGPIKNKM SESPNKTVSE EKAKQYLEEF HQTALEHPEL SELKTVTGTN PVFAGANYAAWAVNVAQVID SETADNLEKT TAALSILPGI GSVMGIADGA VHHNTEEIVA QSIALSSLMVAQAIPLVGEL VDIGFAAYNF VESIINLFQV VHNSYNRPAY SPGHKTQPFL HDGYAVSWNTVEDSIIRTGF QGESGHDIKI TAENTPLPIA GVLLPTIPGK LDVNKSKTHI SVNGRKIRMRCRAIDGDVTF CRPKSPVYVG NGVHANLHVA FHRSSSEKIH

Degree of Labelling Calculated Observed % R_(t) (min) CRM197 58410 n/a 0n/a CRM197 + 1 25 d 59624 59625 14 1.45 CRM197 + 2 25 d 60838 60839 231.45 CRM197 + 3 25 d 62052 62054 35 1.45 CRM197 + 4 25 d 63266 63270 281.45Peptide Mapping Experimental Summary:

Peptide Mapping Digestion: 5 μg modified CRM197 and positive controlCRM197 samples were reduced with 20 mM DTT and digested with 1/30 (w/w)enzyme/protein at 26° C. overnight with trypsin. An aliquot of trypsindigested protein was further digested with GluC enzyme at 1/20enzyme/protein ratio for 4 hr at 26° C.; note all enzymes purchased fromRoche Diagnostics (Gmbh, Germany).

Reverse Phase LC-MS/MS Analysis: Resulting digested peptides wereanalyzed by liquid chromatography electrospray tandem mass spectrometry(LC-ESI MS/MS) on a Thermo LTQ Orbitrap Discovery (Thermo FisherScientific Inc., Waltham, Mass.) coupled to Agilent CapLC (Santa Clara,Calif.). Loaded ˜10-15 pmole of CRM control and modified CRM197 digestson column at 40° C. (Waters Acuity BEH C18, 1.7 μm, 1×100 mm column).Ran 80 min total gradient at 10 μL/min stating at 0-1 min, 4% B,increased to 7% B at 1.1 min, 45% B at 55 min, then 95% B at 63 min,followed by washing and column equilibration. Mass spectrometerparameters included a full scan event using the FTMS analyzer at 30000resolution from m/z 300-2000 for 30 ms. Collision Induced DissociationMS/MS was conducted on the top seven intense ions (excluding 1+ ions) inthe ion trap analyzer, activated at 500 (for all events) signalintensity threshold counts for 30 ms.

Data Analysis and Database Searching: All mass spectra were processed inQual Browser V 2.0.7 (Thermo Scientific). Mascot generic files (mgf)were generated with MS DeconTools (R. D. Smith Lab, PPNL) and searchedusing Mascot V2.3.01 (Matrix Science Inc., Boston, Mass.) databasesearch against the provided protein sequence added to an in-house customdatabase and the SwissProt database (V57 with 513,877 sequences) forcontaminating proteins. Search parameters included: enzyme: semitrypsinor trypsin/Glu-C, allowed up to three missed cleavage; variablemodifications: added expected masses of small molecules (362.147787 Daand 463.206698 Da) to database called “CRM Tgase+alkyne 362 Da mod(CKR), CRM Tgase+alkyne 362 Da mod (N-term), CRM Tgase+azide 463 Da mod(CKR), CRM Tgase+azide 463 Da mod (N-term)”; peptide tolerance: ±20 ppm;MS/MS tolerance: ±0.6 Da. Sequence coverage and small moleculemodification assessments were done on ions scores with >95% confidence.High-scoring peptide ions were then selected for manual MS/MS analysisusing Qual Browser.

Positions at which lysine modification occurs can be determinedaccording to the mapping experiment supra. According to similarconjugations described on CRM197 in US Application No: US 2015/0017192filed on Jul. 11, 2014, we extrapolate that modification occurred onLys37 or Lys39, Lys 33 and Lys440.

Example 26A: Conjugation of Oxytocin Derivative and a Fatty Acid

Step 1: Preparation of Protected Oxytocin on the Resin

The protected from of oxytocin on the resin (26a1) was synthesized byanalogy to Example 21B step 1.

Step 2: Allyl Deprotection, Cyclization, Cleavage from Resin

The protected intermediate (26a1) (0.2 mmol) was taken up in 6 ml of DCMcontaining phenylsilane (1 mmol) and Pd(PPh₃)₄ (0.02 mmol). The resinwas agitated in this solution for 15 minutes and then filtered. Thisprocedure was repeated twice with the phenylsilane/Pd(PPh₃)₄ in DCMsolution. After the last agitation, the resin was filtered and washedwith NMP (3 times), DCM (3 times), 0.5% DIEA/DCM (3 times) and finallyNMP (3 times). The resulting washed resin was dried under vacuum. Aportion of the dried resin (˜0.1 mmol) was taken up in a solution ofPyBOP (0.2 mmol), HOBt (0.4 mmol), and DIEA (0.5 mmol) in 6 mL of NMP.This reaction was agitated at room temperature for 20 hours. The resinwas filtered and washed with EtOAc (3 times) and DCM (3 times). Theresin was then taken up in 4 mL of 95/2.5/2.5 TFA/TIPS/H₂O and agitatedfor 2 hours. The TFA/TIPS/H₂O solution was then filtered into cold(<−20° C.) Et₂O to precipitate the cleaved peptide. Aftercentrifugation, the Et₂O was decanted and the off-white residue waswashed with Et₂O and centrifuged again. The resulting off-white solidwas dried under N₂ overnight. This solid was purified on mass triggeredprepatory HPLC (Waters Autopure HPLC System; Sunfire C18 30×50 mm 5 umcolumn; mobile phase: 7.5-20% ACN in Water, 5 min gradient, 75 mL/min,modified with 0.1% TFA). Fractions corresponding to Intermediate (26a2)were combined, frozen, and lyophilized to a white solid (13.5 mg, 14%).HRMS—Analytic Method G: Rt=0.90 mins, MS m/z 988.5225 [M+H]+.

Step 3: Conjugation to the Fatty Acid

To a solution of Intermediate (26a2) (2.82 μmol) in 0.5 mL of pH=6.40phosphate buffer was added a solution of I-37 (8.39 μmol) in 0.5 ml ofpH=6.40 phosphate buffer. The reaction stirred at room temperature for18 hours. The reaction mixture was then filtered through a 4.5 μm fritand purified on mass triggered prepatory HPLC (Waters Autopure HPLCSystem; Sunfire C18 30×50 mm 5 um column; mobile phase: 45-70% ACN inWater, 5 min gradient, 75 ml/min, modified with 0.1% TFA). Fractionscorresponding to the Oxytocin-FA Conjugate (26A) were combined, frozen,and lyophilized to a white solid (1.71 mg, 24%). LCMS-Analytic Method G:Rt=3.07 mins, MS m/z 2540.5 [M+H]+.

Example 26B: Conjugation of Oxytocin Derivative and a Fatty Acid

The above example (26B) can be prepared according to the step 3 ofExample 26A described above by reacting: (SEQ. ID. NO: 33),*Butyrate-Tyr-Ile-Gln-Asn-Cys*-Gly(N—CH2CH2CH2NH2)-Leu-Gly-NH2*=sulfidebond (26b1):

The cyclic peptide (26b1) was prepared by adapting the procedure ofExample 41 disclosed in Wisniewski et al, J. Med. Chem. (2014), 575306-5317 which is incorporated by reference herein.

The peptide was synthesized manually on 1 mmol Fmoc-Rink Amide AMS resinvia Fmoc chemistry. Protecting groups used for amino acids are: t-Butylgroup for Tyr, Trt group for Gln and Asn. Two unusual amino acids,Fmoc-Cys(CH₂CH₂CH₂CO₂Allyl)-OH and Fmoc-[Na—CH₂CH₂CH₂NH(Boc)]Gly-OH,were used. The peptide chain was assembled on resin by repetitiveremoval of the Fmoc protecting group and coupling of protected aminoacid (3 eq) in DMF. HBTU and HOBt (3 eq: 3 eq) were used as couplingreagent; and N-methylmorpholine (NMM, 6 eq) was used as base. 20%piperidine in DMF (3 times of resin volume) was used as de-Fmoc reagent.Resin was washed by DMF (3 times of resin volume) after each couplingand de-Fmoc; Ninhydrin test was performed after each coupling to checkthe coupling efficiency. Following the removal of the last Fmocprotecting group, the resin was washed with ethyl ether and dried undervacuum. The selective on-resin removal of allyl ester protecting groupwas performed by Pd(PPh3)4/5,5-dimethyl-1,3-cyclohexandione (1 eq/10 eq)in DCM/THF (1/1, 10 times of resin volume) for 3 hours. The resin waswashed with DMF (3×) followed by 0.5% sodium diethyl dithiocarbamate inDMF (5×). In the final, on-resin cyclization was performed by HCTU/NMM(3 eq/6 eq) in DMF. The resin was washed and dried under vacuum to yield3.2 grams of peptide resin which was treated with 32 mlTFA/TIS/DOT/H₂O(92.5/2.5/2.5/2.5, v/v) for 3 hours at room temperatureto remove the side chain protecting groups and cleave the peptide fromthe resin. Crude peptide was precipitated from cold ether then collectedby filtration and dried under high vacuum. Yield: 1.15 g (116%).

All crude of the peptide was purified on 2-inch C18 column with TFAbuffer (buffer A, 0.1% TFA in water; buffer B, acetonitrile). Pooledfractions with purity >95% were lyophilized to dry. 84 mg of finalpeptide was obtained (TFA salt). MS: 991.6 [M+H]⁺, HPLC ret time 9.49min (method: flow rate 1.2 mL/min; Buffer A: 0.1% TFA in water; BufferB: 0.1% TFA in acetonitrile; room temperature; column: Discovery, C18,4.6 mm×250 mm, 5 micro; Gradient (linear) 15%-35% buffer B in 20 mins;injection volume 0.02 mL)

Example 27: Agouti-Related Protein (AgRP)-Fatty-Acid Conjugate

AqRP(83-132)-FA Conjugates:

Example 27A: Mono Fatty Acid Conjugate of AgRP (AgRP+1FA) Wherein theFatty Acid is Attached to the N-Terminus of AgRP Via a Linker (PEG)

wherein AgRP(83-132) has the following sequence:Ser-Ser-Arg-Arg-Cys-Val-Arg-Leu-His-Glu-Ser-Cys-Leu-Gly-Gln-Gln-Val-Pro-Cys-Cys-Asp-Pro-Cys-Ala-Thr-Cys-Tyr-Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys-Tyr-Cys-Arg-Lys-Leu-Gly-Thr-Ala-Met-Asn-Pro-Cys-Ser-Arg-Thr(SEQ. ID. NO: 21); which contains 5 disulfide bridges at positionsC87&C102, C94&C108, C101&C119, C105&C129, C110&C117 Bridges.

Example 27B: Di-Fatty Acid Conjugate of AgRP(83-132) (AgRP+2 FA) WhereinOne Fatty Acid is Attached to the N-Terminus of AgRP (i.e. Serine 83)Via a Linker (PEG) and the Other Fatty Acid is Attached to the SideChain of Lysine at Position 121 Via a PEG Linker

To 0.90 ml of a 10 mg/ml solution of AgRP(83-132) (available from R&DSystems™) in pH 4.5 citrate buffer (9 mg, 1.585 μmol) was added 0.80 mlof pH=4.43 acetate buffer followed by a 1.30 ml of a 10 mg/ml solutionof I-37 in H₂O (13 mg, 7.79 μmol). The reaction stirred at roomtemperature for 16 hours. HRMS (QT2) showed both AgRP+1FA, m/z 7226.3[M+H]⁺ at 1.89 min, and AgRP+2FA, m/z 8778.4 [M+H]⁺ at 2.41 min,present. The reaction was filtered through a 4.5 μm frit, combined witha second reaction ran as above (0.881 μmol AgRP, 2.64 μmol I-37), andpurified on prepatory HPLC (Waters Autopure HPLC System; Waters ProteinBEH C4 Column, 300 Angstrom, 5 um, 10×250 mm; mobile phase: 20-80% ACNin Water, 11 min gradient, 10 mL/min, modified with 0.1% TFA; run time:15 min; fraction collection: UV 210 nm). Fractions corresponding toAgRP+1FA and AgRP+2FA were isolated, frozen, and lyophilized to give theTFA salts of AgRP+1FA (27A) and AgRP+2FA (27B) as white solids (3.24 mg,16% AgRP+1FA; 2.26 mg, 9% AgRP+2FA) LCMS-Analytic Method G: (AgRP+1FA)Rt=1.91 mins, MS m/z 7226.4 [M+H]+; (AgRP+2FA) Rt=2.43 mins, MS m/z8778.4 [M+H]⁺.

Labeling Experiment to Determine Position of Attachment of the FattyAcid.

Labeling at N-terminal Ser residue was confirmed by digesting thereaction mixture with Asp-N (Promega) according to manufacturerprotocol. All peptide mapping assays were achieved using a Thermo DionexUltimate 3000 LC coupled with a Bruker Maxis Impact Q-TOF massspectrometer. The separation was performed on an ACQUITY UPLC BEH130 C18column (2.1×150 mm, 1.7 μm, Waters) kept at 40° C. Flow rate was 0.1mL/min with 0.1% FA in water as mobile phase A and 0.1% FA inacetonitrile as mobile phase B.

A solution of Asp-N (Promega Part #V162A) was reconstituted in 20 uL ofHPLC/MS water (0.1 μg/μL). Around 10 μg of sample was diluted to a finalvolume of 25 μL in 6 M urea, 10 mM dithiothreitol, 5 mM EDTA, and 50 mMTris_HCl (pH=8.0). After reduction and alkylation, solutions werediluted six times with 50 mM Tris_HCl (pH=8.0), proteolysis was thenperformed with an additional 1 micrograms of Asp-N. The digests tookplace overnight at 37 degrees Celsius. LCMS analysis indicated thatcleavage had occurred at the N-terminal D positions of wild AgRP andmodified AgRP with one addition of fatty acid on each fragment as showedin the following table.

Expected Observed peptide sequence position mass RT m/z m/z ChargeSSRRCVRLHESCLGQQVPCC A(1-20) 2488.13 8.1 623.04 623.03 4 (SEQ. ID. NO:22) DPCATCYCRFFNAFCYCRKL A(21-50) 3783.58 10.1 757.72 757.72 5GTAMNPCSRT (SEQ. ID. NO: 23) Modified SSRRCVRLHESCLGQQVPCC A(1-20)4040.12 17.4 1011.04 1011.03 4 (SEQ. ID. NO: 22) + faDPCATCYCRFFNAFCYCRKL A(21-50) 5335.56 18.1 1068.12 1068.12 5 GTAMNPCSRT(SEQ. ID. NO: 23) + fa

Example 28 (28A, 28B and 28C) Relates to Conjugates of hFGF23 Variant

FGF23 Variants Used

The sequence of a human FGF23 variant, used in and designated in thisexample as “hFGF23 (R179Q)”, ““hFGF23 R179”” or simply “hFGF23”, isprovided at SEQ ID NO: 10. This FGF23 variant lacks the signal peptide,but has a restored M at position 1, and has a mutation at R179 (R179Q).A conjugate comprising a fatty acid described herein was prepared withthis FGF23 peptide (Example 28C) and shown to retain at least one FGF23activity in table 8.

Two other variants of human FGF23 were also used in this example(Examples 28A and 28B) to construct conjugates with fatty acidsdisclosed herein. Like the peptide of SEQ ID NO: 10, these lack theFGF23 signal peptide and have a mutation at R179, but have one or moreadditional mutations, but retain at least one FGF23 activity. These twoFGF23 variants are used in and both designated in this example as a“hFGF23-variant” or “FGF23 variant” or the like.

The methods of producing conjugates described herein can be used withother FGF23 peptides, including FGF23, or a homolog, variant, fragment,or modified form thereof.

Protocol for FGF23 Variants Production

Transformation:

The hFGF23 (R179) polypeptide were made by transient transfection of thepET28c-hFGF23 R179Q expression plasmid into BL21(DE3) competent cells,incubating on ice for 30 min, heat shocking at 42° C. for 45 sec, addingSOC media and incubating the bacteria in a 37° C. shaker for one hour.Thereafter, the bacterial culture was spread onto LB plates containingKanamycin and incubated overnight at 37° C. Isolated colonies weretransferred into LB media containing Kanamycin, incubated overnight at37° C. with shaking and 25 mL aliquots transferred into new LB mediacontaining Kanamycin and shaken at 37° C. for about 2.5 hours. When thecells were of sufficient density (OD of ˜0.6), 1M IPTG was added to eachculture with continued shaking at 37° C. to induce expression of thepolypeptide. After four hours the cells were pelleted by centrifugationat 6000 rpm for 10 min and the pellet frozen down at −20° C.Subsequently, the pellet was re-suspended in lysis buffer (50 mM Tris,pH 8, 100 mM NaCl, 0.1% Triton X-100) and the cells lyzed using amicrofluider. 10 mg lysozyme and 10 μL DNase (1 unit per mL, Invitrogen)were added per 100 mL of lysis mix and incubated at room temperature for30 min, then spun down at 8000 rpm at 4° C. for 20 min, washed withthree changes of 100 mL of lysis buffer and spinning, and the fourthtime with lysis buffer without Triton X-100. The pellet (of inclusionbodies) from the final spin was immediately solubilized in 50 mM Tris,pH 7.4, 6 M guanidine, 10 mM DTT, and the protein concentrationdetermined and adjusted to 1 mg/ml before refolding.

Protein Refolding:

To refold the protein, 368 mg of reduced glutathione (GSSH) and 74 mg ofoxidized glutathione (GSSG) were added to each 400 ml of solubilizedinclusion body. The protein was dialyzed overnight at 4° C. against 4 Lof 50 mM Tris, pH 8.0 and 250 mM of Arginine. Then 2L of dialysis bufferwas removed and replaced with 2 L of water and dialysis continued foranother 8 hours. The dialysis buffer was then changed to 20 mM Tris,pH8.0, 50 mM NaCl, 25 mM Arginine, and dialysis continued overnight.

Protein Purification:

To purify the protein, the dialyzed mix was spun down at 12000 rpm for30 min, the supernatant loaded onto Heparin-Sepharose columnequilibrated with the final dialysis buffer, and the column washed with20× bed volume of 1×PBS. The refolded protein was eluted with 1×PBSsupplemented with 0.5M NaCl, the purity of the protein assessed bySDS-PAGE gel, and protein concentration measured by its OD at 280 nmwavelength.

Example 28A: Conjugation of hFGF23 Variant with a Fatty Acid

wherein the —NH₂ in hFGF23varian-NH₂— means the amino functionality of alysine residue.

Step 1: Intermediate 28a

2,5-dioxopyrrolidin-1-yl5-amino-2-(((benzyloxy)carbonyl)amino)-5-oxopentanoate (0.5 g, 1.325mmol) was dissolved in DMF (Volume: 9.96 ml) and tert-butyl(2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (0.503 ml, 2.120 mmol) wasadded. DIPEA (0.274 g, 2.120 mmol) was added to the mixture and thereaction was stirred at r.t. for 2 hours at which point LCMS analysisshowed formation of desired product and consumption of ZQ-NHS startingmaterial (Method A, R_(t)=0.98 min, M+H 511.4). The reaction mixture waspoured into DCM (100 mL) and washed with ice water (3×50 mL). Theorganic layer was dried over Na₂SO₄, filtered and concentrated to give1.14 g of yellow oil. LCMS analysis indicated presence of desiredproduct (Method M, Rt=1.82 min, M+H 511.4, 1.14 g). Material was carriedon to next step without further purification.

Step 2: Intermediate 28b, benzyl(5-amino-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1,5-dioxopentan-2-yl)carbamate

Trifluoroacetic acid (10 mL, 2.233 mmol) was added to Intermediate 28a(1.14 g, 2.233 mmol) and stirred at r.t. for about one hour. LCMSanalysis showed full conversion of starting material to desired product(Method A, R_(t)=0.56 min, M+H 411.3). The reaction mixture was taken upin DCM (30 mL) and concentrated to an oil twice. The oil was dilutedwith 1 mL ACN and 1 mL MeOH and purified by MS-triggered HPLC (MethodN). Fractions with desired mass were pooled, frozen and lyophilized toafford Intermediate 28b as a white powder (Method O, R_(t)=0.22 min, M+H411.3, 160 mg, 18%)

Step 3: Intermediate 28c

Intermediate 28b (19.69 mg, 0.048 mmol) was dissolved in DMF (0.5 mL)and added to a solution of intermediate 37 (50 mg, 0.030 mmol) in DMF(1.0 mL). 3 drops of DIPEA was added and the reaction stirred at r.t.for 2 hours at which point LCMS analysis showed complete conversion toproduct (Method B, R_(t)=1.22 min, M+H+2/2 982.9). The reaction mixturewas loaded on to a 20 g C-18 column for reverse phase chromatography.Using a solvent gradient from 100% Water (0.1% TFA) to 100% MeCN over a20 minute period, fractions collected and analysed by LCMS. Fractionswith desired mass were combined, frozen and lyophilized overnight toafford Intermediate 28c as a clear, colorless oil (Method C, R_(t)=1.21min, M+H+2/2 982.9, M+H+3/3 655.5, 15.3 mg, 26%). Provisionallyinterpreted ¹H-NMR indicates the presence of the amide bond formed at6.29 ppm (1H, br m). ¹H NMR (400 MHz, Chloroform-d) δ 7.52 (s, 1H), 7.35(d, J=3.3 Hz, 5H), 5.10 (s, 2H), 4.30 (s, 1H), 3.77 (t, J=5.8 Hz, 2H),3.69-3.49 (m, 94H), 3.46 (s, 4H), 2.59 (s, 3H), 2.32 (t, J=7.2 Hz, 25H),2.08-1.94 (m, 4H), 1.79-1.65 (m, 2H), 1.65-1.52 (m, 2H), 1.40-1.06 (m,31H), 0.94-0.82 (m, 3H).

Step 4: hFGF23-Variant+Intermediate 28c

For this step, a human FGF23 (hFGF23) variant (“hFGF23-variant”) wasused, which lacks the signal peptide, but has one or more mutationsrelative to SEQ ID NO: 8 but retains at least one FGF23 activity, andwherein the “—NH₂” in “FGF23-variant-NH₂” indicates the aminofunctionality of a lysine residue.

A 50 mg/mL solution of TGase in 30 mM MES pH 6 and an 8 mg/mL solutionof Intermediate 28c in 30 mM MES pH 6 buffer were prepared. The fattyacid solution turned cloudy in MES pH 6. To hFGF23-variant (0.3 mg/mL in30 mM MES pH 6, 7.5 ml, 0.088 μmol) was added Intermediate 28c (217 μl,0.883 μmol) followed by TGase (33.5 μl, 0.044 μmol). The reaction wasmixed at r.t. for 18 hours and an additional 217 μL of Intermediate 28cwas added. The reaction mixed at r.t. for 18 hours and an additional 217μL of Intermediate 28c was added. The reaction mixed at r.t. for 18hours and an additional 108.5 μL of Intermediate 28c was added. Thereaction was mixed at r.t. for 4 hours at which point LCMS analysisshowed complete conversion of starting material (Method P, R_(t)=1.55min, M+H 27432). The reaction mixture was divided between two 4 mL 10kDa MWCO Amicon centrifugal filters and buffer exchanged 3× with 30 mMMES pH 6 buffer, then concentrated to 1.5 mL. Material was stored in therefrigerator overnight. Some solid had settled in the bottom of thetube. Concentration of supernatant was measured by A280 (18730 cm-1M-1,25485 g/mol) to be 0.43 mg/mL (27%).

Example 28B: Conjugation of hFGF23-Variant with a ZQG-PEG11-Fatty Acid

For this example, a conjugate was prepared comprising a fatty acid and aFGF23 variant which lacks the signal peptide and has one or moremutations relative to SEQ ID NO: 8, but the variants retains at leastone FGF23 activity.

wherein the —NH₂ in hFGF23-variant-NH₂ means the amino functionality ofa lysine residue.

An 8 mg/mL solution of intermediate 25d was prepared in 100 mM pH 8 trisbuffer. A 50 mg/mL solution of TGase was prepared in H₂O. To a solutionof hFGF23-variant (6.5 mL, 0.090 μmol) was added intermediate 25d (0.207mL, 1.343 μmol) followed by TGase (0.136 mL, 0.179 μmol). The reactionwas mixed at r.t. for three days and 90% conversion to +1 species wasobserved (LCMS Method Q, R_(t)=7.24 min, M+H 26616). The reactionmixture was exchanged into PBS 1× buffer using 10 kDa MWCO Amiconcentrifugal filters by diluting and concentrating the reaction 6 timesto a volume of 2 mL. The concentration was measured by A280 (18730cm-1M-1, 26617 g/mol) to be 0.125 mg/mL (LCMS Method Q, R_(t)=7.24 min,M+H 26616).

Example 28C: Conjugation of h-FGF23 R179+ZQG-PEG11-Fatty Acid

For this example, a conjugate was prepared using the FGF23 variant“hFGF23 R179”. This lacks the signal peptide and has a mutation at R179.The sequence is provided as SEQ ID NO: 10.

Calculated Mass: 25463

An 8 mg/mL solution of intermediate 25d was prepared in 100 mM pH 8 trisbuffer. A 50 mg/mL solution of TGase was prepared in H₂O. To a solutionof hFGF23 R179 (2.50E+04 μl, 0.393 μmol) was added intermediate 25d (750μl, 4.87 μmol) followed by TGase (597 μl, 0.785 μmol). The reaction wasmixed at r.t. for two days at which point LCMS analysis showed completeconversion of starting material (Method P, R_(t)=1.58 min, M+H 26674).The reaction mixture was purified via ion exchange chromatography togive the +1 conjugate (Method R, R_(t)=3.87 min, M+H 26674):

wherein the —NH₂ in hFGF23 R179-NH₂ means the amino functionality of alysine residue.

Examples 29A and 29B Relate to Conjugates of Serelaxin Example 29A:Serelaxin-Fatty Acid Conjugate (+1 Fatty Acid Conjugate)

Serelaxin+ZQG-PEG₁₁-Fatty Acid (Example 25d):

Serelaxin Sequence:

DSWMEEVIKLCGRELVRAQIAICGMSTWSCFRALSRKTCGVHCCKNALASYLE (SEQ. ID. NO: 24);and

—NH2 in “serelaxin-NH2” means the reactive amino functionality at theside chain of lysine K17 as evidenced by the mappin experiment below; MW5963 g/mol.

An 8 mg/mL solution of ZQG-PEG₁₁-fatty acid (Example 25d) was preparedin 100 mM tris pH 8 buffer. A 50 mg/mL solution of mTGase was preparedin H₂O. To a solution of serelaxin (211 μL, 0.168 μmol) in 100 mM trispH 8 (1.018 mL, 0.5 mg/mL reaction) was added Example 25d (516 μl, 3.35μmol) followed by mTGase (Ajinomoto, 255 μl, 0.335 μmol). The reactionwas mixed at 37° C. for 3 days and then an additional 100 μL ofZQG-PEG₁₁-fatty acid was added. The reaction was mixed at 37° C. for 18hours and then exchanged into PBS 1× buffer using 10 kDa MWCO Amiconcentrifugal filter by diluting and concentrating the reaction 5 times toa volume of 0.7 mL. The material was purified via Method K and thefractions with the desired material were pooled, frozen and lyophilizedto give a white powder. The material was dissolved in 1 mL 30 mM NaOAcbuffer pH 5 and concentration was measured by A280 (5969 cm-1M-1, 7178g/mol) to be 0.25 mg/mL (25%). LCMS Method L: R_(t)=1.65 min; MS [M+1+1FA]: observed: 7180, calculated: 7178.

Experimental Procedure for Serelaxin Mapping

Sample Proteolysis

Approximately 10 μg of protein was dissolved to a final volume of 25 μLin 6 M urea, 10 mM dithiothreitol, 5 mM EDTA, and 50 mM Tris_HCl(pH=8.0) and maintained at 37° C. for 1 hour to reduce disulfide bonds.Iodoacetamide (500 mM, 1 uL) was added to alkylate free thiols and thesolution was allowed to stand at room temperature for 1 hour in thedark. The solution was then diluted 6× with 50 mM Tris_HCl (pH=8.0),LysC (1 ug, Promega V107A) was added and the solution was maintained at37° C. overnight to digest the protein. Formic acid (98%, 2 uL) wasadded to quench proteolysis and the resultant peptide mixture wasanalyzed by LC-MS/MS.

LC-MS/MS Analysis

Peptide mapping was done using a Thermo Dionex Ultimate 3000 HPLCcoupled with a Bruker Maxis Impact Q-TOF mass spectrometer. The MS wascontrolled using Bruker Compass v. 1.7 and Bruker otofControl v. 3.4software, with instrument parameters set as follows: mass range300-2,000 Da; spray voltage 4.0 kV; capillary temperature 200° C.;drying gas flow 5.0 L/min. Fractionation was done with a Waters ACQUITYUPLC BEH130 C18 column (2.1×100 mm, 1.7 μm) maintained at 40° C. Mobilephases were 0.1% formic acid in water and acetonitrile, respectively,and flow rate was 100 uL/min. The gradient used was 0-2 min, 2% B; 2-3min, 2%-8% B; 3-10 min, 8%-29% B; 10-14 min, 29%-33% B; 14-16 min,33%-37% B; 16-20 min, 37%-73% B; 20-22 min, 73%-95% B; 22-25 min, 95% B;25-26 min, 95%-2% B; 26-30 min, 2% B. Data processing was done usingBruker DataAnalysis v. 4.2.

The mapping experiment indicated that the fatty acid addition toserelaxin occurs primarily at Lysine K17 based on the peptide assignedas [CCHVGCTK₁₇(fa)RSLARFC-2H₂O; SEQ. ID. NO: 34], mass: 3088.56 Da,Charge: 5, Rt=13.4 min, Observed m/z 618.71, Expected m/z 618.72.

Example 29B: Serelaxin-Fatty Acid Conjugate (Mixture of +1FA, +2FA and+3FA Conjugate as Described Below)

wherein the —NH₂ in “serelaxin-NH₂” means the reactive aminofunctionality at the side chain of a lysine.Serelaxin+Intermediate 37: Example 29B was Tested as the Mixture Below

Degree of Labelling Calculated Observed % serelaxin 5963 5964 3serelaxin + 1 FA 7517 7516 19 serelaxin + 2 FA 9071 9069 52 serelaxin +3 FA 10625 10622 25

Sequence:

DSWMEEVIKLCGRELVRAQIAICGMSTWSCFRALSRKTCGVHCCKNALASYLpE (SEQ. ID. NO: 24)

A 10 mg/mL solution of fatty acid intermediate 37 was prepared in H₂O.To a solution of serelaxin (105 μL, 0.084 μmol, 4.75 mg/mL) in 30 mMNaOAc buffer pH 4 (755 μL) was added fatty acid intermediate 37 (140 μL,0.839 μmol). The reaction was mixed at r.t. for 16 hours at which pointLCMS analysis showed 90% conversion of starting material. An additional70 μL of intermediate 37 was added and the reaction mixed for 16 hoursat r.t. at which point MALDI analysis indicated >95% conversion. Thesolution was exchanged into PBS 1× buffer using 3 kDa MWCO Amiconcentrifugal filter by diluting and concentrating the reaction 4 times toa volume of 0.2 mL. The concentration was measured by A₂₈₀ (5969 M-1cm-1; 9068 g/mol) to be 2.61 mg/mL. LCMS Method L: R_(t)=1.56 min; MS[M+1 +1 FA]: observed: 7516, calculated: 7517; R_(t)=1.65 min; MS [M+1+2 FA]: observed: 9069, calculated: 9071; R_(t)=1.74 min; MS [M+1 +3FA]: observed: 10622, calculated: 10625.

Example 30: Conjugation of M-his-hPIP with Fatty Acid Construct(I-37)—(Mixture of +1 FA Conjugate, and +2 FA Conjugates as DescribedBelow)

M-His-hPIP (29-146) Sequence:

(SEQ ID NO: 13) MHHHHHHQDNTRKIIIKNFDIPKSVRPNDEVTAVLAVQTELKECMVVKTYLISSIPLQGAFNYKYTACLCDDNPKTFYWDFYTNRTVQIAAVVDVIRELGICPDDAAVIPIKNNRFYTIEILKVEExpressed fromExpressed Protein Sequence:

(SEQ. ID. NO: 25) METDTLLLWVLLLWVPGSTGMHHHHHHQDNTRKIIIKNFDIPKSVRPNDEVTAVLAVQTELKECMVVKTYLISSIPLQGAFNYKYTACLCDDNPKTFYWDFYTNRTVQIAAVVDVIRELGICPDDAAVIPIKNNRFYTIEILKVENucleotide Sequence:

(SEQ. ID. NO: 26) GCTAGCCACCATGGAGACTGATACTTTGTTGTTGTGGGTACTGTTGCTTTGGGTGCCCGGTAGTACCGGTATGCATCACCACCACCATCACCAGGACAACACCCGGAAGATCATCATCAAGAACTTCGACATCCCTAAGAGCGTGCGCCCAAACGATGAAGTCACCGCGGTGCTGGCAGTGCAGACTGAGCTGAAGGAGTGCATGGTGGTCAAGACGTACCTGATTTCGTCCATCCCGCTGCAAGGCGCCTTCAACTACAAGTACACTGCCTGCCTCTGTGACGACAACCCCAAGACCTTTTACTGGGACTTCTACACCAATAGAACTGTCCAGATTGCTGCCGTGGTGGATGTGATCAGGGAATTGGGAATTTGCCCCGACGATGCGGCCGTGATTCCGATCAAGAACAACCGCTTCTATACCATCGAGATCCTTAAAGTGGAATGAGA ATTCPIP Expression Vector:

A mammalian expression vector encoding human PIP was generated bystandard cloning methods. A fragment containing the mouse Ig kappa chainsignal sequence followed by a MHHHHH (SEQ. ID. NO: 27) sequence thenmature PIP with 5′-NheI (followed by a Kozak sequence) and 3′-EcoRIsites was codon optimized and synthesized (DNA2.0). This sequence wasthen cloned into unique 5′-NheI and 3′-EcoRI sites of a pcDNA3.1(Invitrogen) based vector downstream of the CMV promoter.

PIP Expression and Purification:

The PIP expression plasmid DNA was transfected into HEK293T cells at adensity of 1×10⁶ cells per ml using standard polyethylenimine methods.500 ml cultures were then grown in FreeStyle 293 Medium (LifeTechnologies) in 3 L flasks for 4 days at 37° C. with a humidifiedatmosphere of 8% CO₂. PIP protein was purified from clarifiedconditioned media.

wherein M-His-PIP has SEQ ID NO: 12 and “M-his” is MHHHHHH (SEQ. ID. NO:16).Conjugation:

A 10 mg/mL solution of fatty acid-linker construct #1 was prepared inH₂O. M-His-hPIP (0.700 mL, 0.048 μmol) was diluted with 30 mM NaOAcbuffer pH 4 (619 μL. 0.5 mg/mL reaction) and Intermediate 37 (0.081 mL,0.484 μmol) was added. The reaction was mixed at r.t. for 18 hours atwhich point LCMS analysis showed 70% conversion to +1 (50%) and +2 (20%)products. The material was then exchanged into PBS 1× buffer using 3 kDaMWCO Amicon centrifugal filter by diluting and concentrating thereaction 5 times to a volume of 350 μL. Concentration was measured byA₂₈₀ (13850 cm-1M-1, 14472 g/mol) to be 1.7 mg/mL (70%). LCMS Method L,R_(t)=1.46 min; MS [M+1]: observed: 14472, calculated: 14476. R_(t)=1.57min; MS [M+1 +1FA deglycosylated]: observed: 16025, calculated: 16030.R_(t)=1.69 min; MS [M+1 +2FA deglycosylated]: observed: 17578,calculated: 17584.

M-his-hPIP+Fatty Acid-Linker Construct I-37: Example 30 was Tested asthe Mixture Below

Degree of Labelling Calculated Observed % M-His-PIP 14476 14472 4M-His-PIP + glycosylation 18139 30 M-His-PIP + 1 FA 16030 16031 7M-His-PIP + 1 FA + glycosylation 19692 37 M-His-PIP + 2 FA 17584 1757815 M-His-PIP + 2 FA + glycosylation 21242 7Experimental procedure for PIP mappingSample Proteolysis

Approximately 10 μg of protein was dissolved to a final volume of 25 μLin 6 M urea, 10 mM dithiothreitol, 5 mM EDTA, and 50 mM Tris_HCl(pH=8.0) and maintained at 37° C. for 1 hour to reduce disulfide bonds.Iodoacetamide (500 mM, 1 uL) was added to alkylate free thiols and thesolution was allowed to stand at room temperature for 1 hour in thedark. The solution was then diluted 6× with 50 mM Tris_HCl (pH=8.0),LysC (1 ug, Promega) or Trypsin/Lys C mix (1 ug, Promega) was added andthe solution was maintained at 37° C. overnight to digest the protein.Formic acid (98%, 2 uL) was added to quench proteolysis and theresultant peptide mixture was analyzed by LC-MS/MS.

LC-MS/MS Analysis

Peptide mapping was done using a Thermo Dionex Ultimate 3000 HPLCcoupled with a Bruker Maxis Impact Q-TOF mass spectrometer. The MS wascontrolled using Bruker Compass v. 1.7 and Bruker otofControl v. 3.4software, with instrument parameters set as follows: mass range300-2,000 Da; spray voltage 4.0 kV; capillary temperature 200° C.;drying gas flow 5.0 L/min. Fractionation was done with a Waters ACQUITYUPLC BEH130 C18 column (2.1×100 mm, 1.7 μm) maintained at 40° C. Mobilephases were 0.1% formic acid in water and acetonitrile, respectively,and flow rate was 100 uL/min. The gradient used was 0-2 min, 2% B; 2-3min, 2%-8% B; 3-10 min, 8%-29% B; 10-14 min, 29%-33% B; 14-16 min,33%-37% B; 16-20 min, 37%-73% B; 20-22 min, 73%-95% B; 22-25 min, 95% B;25-26 min, 95%-2% B; 26-30 min, 2% B. Data processing was done usingBruker DataAnalysis v. 4.2.

The mapping experiment indicated that the fatty acid addition to MH₆-PIPoccurs preferentially at the N-terminus as evidenced by[fa-MHHHHHHQDNTRK; SEQ. ID. NO: 35], mass: 3265.76 Da, Charge: 4,Rt=23.4 min, Observed m/z: 817.44, Expected m/z: 817.45.

A small degree of fatty acid addition occurs at lysine K42 as evidencedby peptide fragment [SVRPNDEVTAVLAVQTELK(fa)ECMVVK; SEQ. ID. NO: 36],mass: 4366.45 Da, Charge 3, Rt=23.5 min, Observed m/z: 1456.49, Expectedm/z: 1456.49. Addition of the fatty acid at K42 blocks trypsin cleavageadjacent to this lysine, serving to confirm location of the addition.

Example 31: Conjugation of NPFF with Fatty Acid Using Click Chemistry

Step 1: Preparation of NPFF Click Chemistry Handle

Calc. MH+ 1258.8

To a solution of NPFF (Alfa Aesar, J66509, 5 mg, 3.82 μmol) in DMSO (1mL) was added triethylamine (5.32 μl, 0.038 mmol) and then(1R,8S,9r)-bicyclo[6.1.0]non-4-yn-9-ylmethyl (2,5-dioxopyrrolidin-1-yl)carbonate (1.335 mg, 4.58 μmol). The reaction mixture was stirred atroom temperature. Upon completion the reaction mixture was taken on ascrude to the next reaction step.

Step 2: Conjugation

To the crude reaction mixture from Step 1 was added Intermediate 6(I-6). The reaction mixture was shaken at room temperature. Uponcompletion the reaction mixture was purified using reverse phasechromatography (System: Agilent Bioinert SystemDate; Column: WatersProtein BEH C4 Column, 300 Angstrom, 5 um, 10×250 mm; Column for UPLCmethod development is BEH C4m, 300A, 1.7 um, 2.1×50 mm; Mobile Phase:46-56% ACN gradient in 6 min, Modified with 0.1% TFAA: Water, B:Acetonitrile; Flow Rate: 2.0 mL/min; Run time: 15 min; Fractioncollection: UV 210 nm) to afford the titled compound, a white solid as aTFA salt; LCMS: Method S: ELSD: Rt 1.46 mins; MS m/z 928.3 [(M/3)⁺H]⁺

It can be seen that the conjugates of the invention have similar orimproved efficacy as compared to the non-conjugated biomolecule butadditionally the conjugates of the invention have improved plasmastability compared to the non-conjugated biomolecule. The conjugates inthe examples above have been found to have a plasma stability higherthan 5 h, higher than 10 h, higher than 20 h, higher than 30 h, higherthan 40 h, and in some cases higher than 50 h.

Having thus described exemplary embodiments of the present invention, itshould be noted by those of ordinary skill in the art that the withindisclosures are exemplary only and that various other alternatives,adaptations, and modifications may be made within the scope of thepresent invention. Accordingly, the present invention is not limited tothe specific embodiments as illustrated therein.

What is claimed is:
 1. A method of treating or preventing a disease ordisorder selected from metabolic disorders or diseases, type 2 diabetesmellitus, obesity, pancreatitis, dyslipidemia, nonalcoholicsteatohepatitis, insulin resistance, hyperinsulinemia, glucoseintolerance, hyperglycemia, metabolic syndrome, hypertension,cardiovascular disease, atherosclerosis, peripheral arterial disease,stroke, heart failure, coronary heart disease, diabetic complications,chronic kidney disease, neuropathy, and gastroparesis, in a subject inneed thereof, comprising administering to the subject a therapeuticallyeffective amount of a conjugate comprising a biomolecule linked to afatty acid via a linker, wherein the fatty acid has the followingFormula A1:

R¹ is CO₂H; R² and R³ are independently of each other H, OH, CO₂H,—CH═CH₂ or —C≡CH; and n and m are independently of each other an integerbetween 6 and 30; and wherein the biomolecule is MH(199-308)hGDF15 (SEQID NO: 4), MHA(200-308)hGDF15 (SEQ ID NO: 6), AHA(200-308)hGDF15 (SEQ IDNO: 7), AH(199-308)hGDF15 (SEQ ID NO: 5), MHHHHHHM-hGDF15 (SEQ ID NO:2), MHHHHHH-hGDF15 (SEQ ID NO: 1), or his-hGDF15; or a dimer thereof,wherein his-hGDF15 is hGDF15(197-308) wherein a tag, comprising 1 to 6histidine amino acids and optionally 1 or 2 methionine amino acids, hasbeen added to the N-terminus of hGDF15; or an amide, ester orpharmaceutically acceptable salt thereof.
 2. The method according toclaim 1, wherein the fatty acid is selected from:

wherein Ak³, Ak⁴, Ak⁵, Ak⁶ and Ak⁷ are independently a linear(C₈₋₂₀)alkylene, and R⁵ and R⁶ are independently linear (C₈₋₂₀)alkyl, oran amide, an ester or a pharmaceutically acceptable salt thereof.
 3. Themethod according to claim 1, wherein the fatty acid is selected from:

or an amide, ester or a pharmaceutically acceptable salt thereof.
 4. Themethod according to claim 1, wherein the linker comprises alkyl,alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, polyethyleneglycol, or one or more natural or unnatural amino acids, or combinationthereof, wherein each of the alkyl, alkenyl, cycloalkyl, aryl,heteroaryl, heterocyclyl, polyethylene glycol and/or the natural orunnatural amino acids are optionally combined and linked together orlinked to the biomolecule and/or to the fatty acid moiety via a chemicalgroup selected from —C(O)O—, —OC(O)—, —NHC(O)—, —C(O)NH—, —O—, —NH—,—S—, —C(O)—, —OC(O)NH—, —NHC(O)—O—, ═NH—O—, ═NH—NH— and ═NH—N(alkyl)-;or an amide, ester or a pharmaceutically acceptable salt thereof.
 5. Themethod according to claim 1, wherein the linker comprises an unbranchedoligo ethylene glycol moiety of Formula:

wherein y is 0 to 34; or an amide, an ester or a pharmaceuticallyacceptable salt thereof.
 6. The method according to claim 1, wherein thelinker comprises a heterocyclic moiety of the following Formula:

wherein r is an integer of 0 to 2, and s is an integer of 0 to 3; or anamide, an ester or a pharmaceutically acceptable salt thereof.
 7. Themethod according to claim 1, wherein the linker comprises one or moreamino acids independently selected from histidine, methionine, alanine,glutamine, asparagine, and glycine; or an amide, an ester or apharmaceutically acceptable salt thereof.
 8. The method according toclaim 1, wherein the biomolecule linked to the fatty acid via a linkerhas one of the following Formulae:

wherein in Formulae C and D, both monomeric units of his-hGDF15 or ofhGDF15* are linked to the fatty acid moiety via a linker at theN-terminus of each his-hGDF15 monomeric unit and at the N-terminus ofeach hGDF15* monomeric unit; wherein in Formulae E and F, only one ofthe monomeric unit of his-hGDF15 or of hGDF15* is linked to the fattyacid moiety via a linker at the N-terminus; wherein in Formulae D and F,hGDF15* is MH(199-308)hGDF15 (SEQ ID NO: 4), MHA(200-308)hGDF15 (SEQ IDNO: 6), AHA(200-308)hGDF15 (SEQ ID NO: 7), AH(199-308)hGDF15 (SEQ ID NO:5), MHHHHHHM-hGDF15 (SEQ ID NO: 2), or MHHHHHH-hGDF15 (SEQ ID NO: 1);wherein in Formulae C and E, his-hGDF15 is hGDF15 (197-308) wherein atag, comprising 1 to 6 histidine amino acids and optionally 1 or 2methionine amino acids, has been added to the N-terminus of hGDF15; andwherein in Formulae C, D, E, and F, s is an integer between 20-30; andthe line between the 2 monomeric units of his-hDGF15 or the 2 monomericunits of hGDF15* represents a disulfide bond.
 9. The method according toclaim 8, wherein the method comprises administering to the subject atherapeutically effective amount of a mixture comprising the conjugateaccording to claim 8 having Formula C and the conjugate according toclaim 8 having Formula E or a mixture comprising the conjugate accordingto claim 8 having Formula D and the conjugate according to claim 8having Formula F.
 10. The method according to claim 8, wherein thebiomolecule linked to a fatty acid via a linker is of Formula G or ofFormula H:

wherein AHA-hGDF15 is AHA(200-308)hGDF15 (SEQ ID NO: 7) and the fattyacid is linked via a linker at the N-terminus of one AHA-hGDF15monomeric unit in Formula H or via a linker at both the N-terminus ofeach of the two AHA-hGDF15 monomeric units in Formula G, and wherein theline between the two AHA-hGDF15 units represents a disulfide bond. 11.The method according to claim 10, wherein the method comprisesadministering to the subject a therapeutically effective amount of amixture comprising the conjugate according to claim 10 having Formula Gand the conjugate according to claim 10 having Formula H.
 12. The methodaccording to claim 1, wherein the fatty acid moiety is attached to theN-terminus of the biomolecule via a linker; or an amide, an ester or apharmaceutically acceptable salt thereof.
 13. The method according toclaim 1, wherein the conjugate has a plasma stability half-life of morethan 10 hours, more than 20 hours or more than 30 hours.
 14. The methodaccording to claim 1, where the improvement of plasma stability comparedto the non-conjugated biomolecule is 2 fold, 5 fold, 10 fold, 20 fold,30 fold, 40 fold, 50 fold, or 75 fold.
 15. The method according to claim1, further comprising administering one or more therapeutically activeco-agents.
 16. The method according to claim 15, wherein the co-agent isselected from antidiabetic agent, hypolipidemic agent, anti-obesityagents, anti-hypertensive agents, and agonists of peroxisomeproliferator-activator receptors.
 17. The method according to claim 16,wherein the co-agent is selected from insulin, insulin derivatives andmimetics; insulin secretagogues; glyburide, glimepiride; insulinotropicsulfonylurea receptor ligands; thiazolidinediones, pioglitazone,balaglitazone, rivoglitazone, netoglitazone, troglitazone, englitazone,ciglitazone, adaglitazone, darglitazone, Cholesteryl ester transferprotein (CETP) inhibitors, GSK3 (glycogen synthase kinase-3) inhibitors;Retinoid X receptor ligands; sodium-dependent glucose cotransporterinhibitors; glycogen phosphorylase A inhibitors; biguanides;alpha-glucosidase inhibitors, GLP-1 (glucagon like peptide-1), GLP-1analogs, GLP-1 mimetics; DPPIV (dipeptidyl peptidase IV) inhibitors,3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors;squalene synthase inhibitors; FXR (farnesoid X receptor), LXR (liver Xreceptor) ligands; cholestyramine; fibrates; nicotinic acid, aspirin;orlistat or rimonabant; loop diuretics, furosemide, torsemide;angiotensin converting enzyme (ACE) inhibitors; inhibitors of theNa-K-ATPase membrane pump; neutralendopeptidase (NEP) inhibitors;ACE/NEP dual inhibitors; angiotensin II antagonists; renin inhibitors;0-adrenergic receptor blockers; inotropic agents, dobutamine, milrinone;calcium channel blockers; aldosterone receptor antagonists; aldosteronesynthase inhibitors; fenofibrate, pioglitazone, rosiglitazone,tesaglitazar, BMS-298585, and L-796449.
 18. A method of treating orpreventing a disease or disorder selected from metabolic disorders ordiseases, type 2 diabetes mellitus, obesity, pancreatitis, dyslipidemia,nonalcoholic steatohepatitis, insulin resistance, hyperinsulinemia,glucose intolerance, hyperglycemia, metabolic syndrome, hypertension,cardiovascular disease, atherosclerosis, peripheral arterial disease,stroke, heart failure, coronary heart disease, diabetic complications,chronic kidney disease, neuropathy, and gastroparesis, in a subject inneed thereof, wherein the method comprises administering to the subjecta therapeutically effective amount of a pharmaceutical compositioncomprising a therapeutically effective amount of a conjugate accordingto claim 1; or a mixture of conjugates comprising the conjugateaccording to claim 10 having Formula G and the conjugate according toclaim 10 having Formula H, or an amide, an ester or a pharmaceuticallyacceptable salt thereof, and one or more pharmaceutically acceptablecarriers.
 19. The method according to claim 11, further comprisingadministering one or more therapeutically active co-agents.
 20. Themethod according to claim 19, wherein the co-agent is selected fromantidiabetic agent, hypolipidemic agent, anti-obesity agents,anti-hypertensive agents, and agonists of peroxisomeproliferator-activator receptors.
 21. The method according to claim 20,wherein the co-agent is selected from insulin, insulin derivatives andmimetics; insulin secretagogues; glyburide, glimepiride; insulinotropicsulfonylurea receptor ligands; thiazolidinediones, pioglitazone,balaglitazone, rivoglitazone, netoglitazone, troglitazone, englitazone,ciglitazone, adaglitazone, darglitazone, Cholesteryl ester transferprotein (CETP) inhibitors, GSK3 (glycogen synthase kinase-3) inhibitors;Retinoid X receptor ligands; sodium-dependent glucose cotransporterinhibitors; glycogen phosphorylase A inhibitors; biguanides;alpha-glucosidase inhibitors, GLP-1 (glucagon like peptide-1), GLP-1analogs, GLP-1 mimetics; DPPIV (dipeptidyl peptidase IV) inhibitors,3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors;squalene synthase inhibitors; FXR (farnesoid X receptor), LXR (liver Xreceptor) ligands; cholestyramine; fibrates; nicotinic acid, aspirin;orlistat or rimonabant; loop diuretics, furosemide, torsemide;angiotensin converting enzyme (ACE) inhibitors; inhibitors of theNa-K-ATPase membrane pump; neutralendopeptidase (NEP) inhibitors;ACE/NEP dual inhibitors; angiotensin II antagonists; renin inhibitors;β-adrenergic receptor blockers; inotropic agents, dobutamine, milrinone;calcium channel blockers; aldosterone receptor antagonists; aldosteronesynthase inhibitors; fenofibrate, pioglitazone, rosiglitazone,tesaglitazar, BMS-298585, and L-796449.
 22. The method according toclaim 11, wherein the mixture is a 1:1 molar ratio of a conjugate ofFormula G and a conjugate of Formula H.
 23. The method according toclaim 1, wherein the biomolecule is MH(199-308)hGDF15 (SEQ ID NO: 4),MHA(200-308)hGDF15 (SEQ ID NO: 6), AHA(200-308)hGDF15 (SEQ ID NO: 7), orAH(199-308)GDF15 (SEQ ID NO: 5); or a dimer thereof.
 24. A method oftreating or preventing a disease or disorder selected from type 2diabetes mellitus, obesity, cardiovascular disease, and heart failure,in a subject in need thereof, comprising administering to the subject atherapeutically effective amount of a conjugate of Formula G or ofFormula H or a mixture of conjugates comprising the conjugate havingFormula G and the conjugate having Formula H:

wherein AHA-hGDF15 is AHA(200-308)hGDF15 (SEQ ID NO: 7) and the fattyacid is linked via a linker at the N-terminus of one AHA-hGDF15monomeric unit in Formula H or via a linker at both the N-terminus ofeach of the two AHA-hGDF15 monomeric units in Formula G, and wherein theline between the two AHA-hGDF15 units represents a disulfide bond, or anamide, ester or pharmaceutically acceptable salt thereof.
 25. The methodaccording to claim 24, wherein the disease or disorder is type 2diabetes mellitus.
 26. The method according to claim 24, wherein thedisease or disorder is obesity.
 27. The method according to claim 24,wherein the disease or disorder is cardiovascular disease.
 28. Themethod according to claim 24, wherein the disease or disorder is heartfailure.
 29. A method of treating or preventing a disease or disorderselected from type 2 diabetes mellitus, obesity, cardiovascular disease,and heart failure, in a subject in need thereof, wherein the methodcomprises administering to the subject a therapeutically effectiveamount of a pharmaceutical composition comprising a conjugate of FormulaG or of Formula H or a mixture of conjugates comprising the conjugatehaving Formula G and the conjugate having Formula H:

wherein AHA-hGDF15 is AHA(200-308)hGDF15 (SEQ ID NO: 7) and the fattyacid is linked via a linker at the N-terminus of one AHA-hGDF15monomeric unit in Formula H or via a linker at both the N-terminus ofeach of the two AHA-hGDF15 monomeric units in Formula G, and wherein theline between the two AHA-hGDF15 units represents a disulfide bond, or anamide, ester or pharmaceutically acceptable salt thereof, and one ormore pharmaceutically acceptable carriers.
 30. The method according toclaim 29, wherein the disease or disorder is type 2 diabetes mellitus.31. The method according to claim 29, wherein the disease or disorder isobesity.
 32. The method according to claim 29, wherein the disease ordisorder is cardiovascular disease.
 33. The method according to claim29, wherein the disease or disorder is heart failure.